HK1112834A - Molecular antigen array - Google Patents

Description

Molecular antigen array

the application is a divisional application of an application with the application date of 2002, 1/21, the application number of 02803869.X and the name of 'molecular antigen array'.

Background

Technical Field

The present invention relates to the fields of molecular biology, virology, immunology and medicine. The present invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array. The invention also provides a method of producing an antigen or antigenic determinant in an ordered and repetitive array. The ordered and repetitive antigens or antigenic determinants are useful in the production of vaccines for the treatment of infectious diseases, the treatment of allergies, and as medicaments for the prevention or treatment of cancer, effective induction of self-specific immune responses, particularly antibody responses.

Background

WO 003227 describes compositions and methods for producing ordered and repetitive antigen or antigenic determinant arrays. These compositions are useful in the manufacture of vaccines for the prevention of infectious diseases, the treatment of allergies and the treatment of cancer. These compositions comprise a core particle, such as a virus or virus-like particle, to which at least one antigen or antigenic determinant is bound by at least one non-peptide bond, resulting in an ordered and repetitive antigen array.

Virus-like particles (VLPs) find application in the field of vaccine production due to their structural characteristics and non-infectivity. VLPs are supramolecular structures that are built up in a symmetric fashion by many protein molecules of one or more types. They lack the viral genome and are therefore not infectious. VLPs can generally be produced in large quantities by heterologous expression and are easy to purify.

Examples of VLPs include: hepatitis B Virus (Ulrich et al, Virus Res.50: 141-182(1998)), measles Virus (Warnes et al, Gene 160: 173-.

Due to immune tolerance, it is often difficult to elicit an immune response against self molecules. In particular, lymphocytes with self-molecular specificity are often hyporeactive or even anergic if triggered by conventional vaccination strategies.

Amyloid B peptide (A beta)_1-42) Plays a role in neuropathology of Alzheimer's diseaseHas the main function. Regional specific extracellular accumulation of a β peptide is associated with microgliosis, cytoskeletal changes, dystrophic neuritis and synaptic loss. These pathological changes are generally thought to be associated with cognitive deficits that define the disease.

Production of Abeta in a mouse model of Alzheimer's disease_1-42The engineered transgenic animals of (PDAPP-mice) form plaques and neuronal damage in the brain. Recent studies have shown that with A β_1-42Immunization of young PDAPP-mice results in inhibition of plaque formation and associated dystrophic neuritis (Schenk, D. et al, Nature 400: 173-77 (1999)).

In addition, immunization of PDAPP mice that have developed AD-like neuropathologies may also reduce the extent and progression of neuropathology. The immunization protocol for these studies was as follows: peptides were dissolved in aqueous buffer and mixed 1: 1 with complete Freund's adjuvant (initial dose) to give a peptide concentration of 100. mu.g/dose. Subsequent boosts were with incomplete freund's adjuvant. Mice received 11 immunizations within 11 months. Antibody titers above 1: 10000 are achieved and maintained. Thus, immunization may be an effective prophylactic and therapeutic approach for alzheimer's disease.

In another study, peripherally administered anti-a β_1-42Antibodies are capable of crossing the blood-brain barrier, binding to the A β peptide, and inducing clearance of existing amyloid (Bard, F. et al, Nature Medicine 6: 916-19 (2000)). The study adopted anti-Abeta_1-42Or a monoclonal antibody against a synthetic fragment derived from a different region of a β. Thus, induction of antibodies may be considered as a possible treatment for alzheimer's disease.

It has been determined that administration of purified protein alone is generally insufficient to elicit a strong immune response; the isolated antigen must generally be combined with an auxiliary substance known as an adjuvant. Adjuvants protect the administered antigen from rapid degradation and adjuvants can allow low levels of antigen to be released over a long period of time.

As previously mentioned, one of the important events in Alzheimer's Disease (AD) is the deposition of amyloid as an insoluble fibrous mass (amyloid production), forming extracellular neuritic plaques and deposits around the walls of the cerebral vessels (reviewed in Selkoe, D.J, (1999) Nature399, a 23-31). The major component of neuritic plaques and congo red (congophilic) vascular disease is amyloid beta (a β), although these deposits also contain other proteins, such as glycosaminoglycans and apolipoproteins. A β is proteolytically cleaved from a large glycoprotein called Amyloid Precursor Protein (APP), which comprises a 695-152 amino acid isoform with a hydrophobic transmembrane region. A β forms a group of peptides up to 43 amino acids in length that exhibit considerable amino-and carboxy-terminal heterogeneity (truncation) as well as modifications (Roher, a.e., Palmer, k.c., Chau, V. & Ball, m.j. (1998) j.cell biol.107, 2703-. The major isoforms are A.beta.1-40 and 1-42. It is highly prone to form beta sheets that can aggregate into fibrils, ultimately producing amyloid. Recent studies have demonstrated that vaccination-induced reduction of cerebral amyloid deposits leads to an increase in cognitive performance (Schenk, d., barbeur, r., Dunn, w., Gordon, g., Grajeda, h., Guido, t., Hu, k., Huang, j., Johnson-Wood, k., Khan, k., et al (1999) Nature400, 173-.

We have surprisingly found that self-molecules or self-antigens present in highly ordered, repetitive arrays are effective in eliciting self-specific immune responses, particularly antibody responses. Moreover, this response can be induced even in the absence of adjuvants that non-specifically activate antigen presenting cells and other immune cells.

Summary of The Invention

The present invention provides compositions comprising highly ordered, repetitive antigens or antigenic determinant arrays, and methods of production and use thereof. Thus, the compositions of the invention are useful for the production of vaccines for the prevention of infectious diseases, the treatment of allergies and cancer, and the effective induction of self-specific immune responses, in particular antibody responses.

In a first aspect, the present invention provides a novel composition comprising or consisting of: (A) a non-natural molecular scaffold, and (B) an antigen or antigenic determinant. Such non-natural molecular scaffolds comprise or consist of: (i) a core particle selected from: (1) a core particle of non-natural origin and (2) a core particle of natural origin; (ii) an organiser (organosizer) comprising at least one first attachment site, wherein the organiser is linked to the core particle by at least one covalent bond. The antigen or antigenic determinant is an autoantigen or fragment thereof having at least one second attachment site selected from the group consisting of: (i) a non-naturally occurring attachment site for the antigen or antigenic determinant; (ii) the naturally occurring attachment site for the antigen or antigenic determinant. The present invention provides an ordered and repetitive autoantigen array by binding the second attachment site to the first attachment site via at least one non-peptide bond. Thus, through the binding of the first attachment site to the second attachment site, the autoantigen or autoantigenic determinant is bound to the unnatural molecular scaffold, forming an ordered and repetitive antigen array.

In a second aspect, the present invention provides a novel composition comprising or consisting of: (A) a non-natural molecular scaffold, and (B) an antigen or antigenic determinant. Such non-natural molecular scaffolds comprise or consist of: (i) a core particle and (ii) an organizer comprising at least one first attachment site, wherein the core particle is a virus-like particle comprising recombinant proteins or fragments thereof of a bacteriophage, wherein the organizer is linked to the core particle by at least one covalent bond. The antigen or antigenic determinant has at least one second attachment site selected from the group consisting of: (i) a non-naturally occurring attachment site for the antigen or antigenic determinant; (ii) the naturally occurring attachment site for the antigen or antigenic determinant. The present invention provides an ordered and repetitive antigen array by binding the second attachment site to the first attachment site via at least one non-peptide bond.

In a third aspect, the present invention provides a novel composition comprising or consisting of: (A) a non-natural molecular scaffold, and (B) an antigen or antigenic determinant. Such non-natural molecular scaffolds comprise or consist of: (i) a core particle selected from: (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (ii) an formed body having at least one first attachment site, wherein the formed body is linked to the core particle by at least one covalent bond. The antigen or antigenic determinant is an amyloid beta peptide (A beta) _1-42) Or a fragment thereof, having at least one second attachment site selected from the group consisting of: (i) a non-naturally occurring attachment site for the antigen or antigenic determinant; (ii) the naturally occurring attachment site for the antigen or antigenic determinant. The present invention provides an ordered and repetitive antigen array by binding the second attachment site to the first attachment site via at least one non-peptide bond.

In a fourth aspect, the present invention provides a novel composition comprising or consisting of: (A) a non-natural molecular scaffold, (B) an antigen or antigenic determinant. Such non-natural molecular scaffolds comprise or consist of: (i) a core particle selected from: (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (ii) an formed body having at least one first attachment site, wherein the formed body is linked to the core particle by at least one covalent bond. The antigen or antigenic determinant is an anti-idiotypic antibody or anti-idiotypic antibody fragment having at least one second attachment site selected from the group consisting of: (i) a non-naturally occurring attachment site for the antigen or antigenic determinant; (ii) the naturally occurring attachment site for the antigen or antigenic determinant. The present invention provides an ordered and repetitive antigen array by binding the second attachment site to the first attachment site via at least one non-peptide bond.

Other aspects and preferred embodiments and advantages of the invention will become apparent hereinafter, particularly from the detailed description, examples and claims.

In a preferred embodiment of the invention, the core particle is a virus-like particle comprising recombinant proteins of RNA-phages, preferably selected from the group consisting of: a) bacteriophage Q β; b) bacteriophage R17; c) bacteriophage fr; d) phage GA; e) phage SP; f) bacteriophage MS 2; g) bacteriophage M11; h) phage MX 1; i) bacteriophage NL 95; k) bacteriophage f 2; l) bacteriophage PP 7. Most preferred are bacteriophage Q β and bacteriophage fr.

In another preferred embodiment of the invention, the recombinant protein of the RNA-phage comprises a wild-type coat protein.

In yet another preferred embodiment of the invention, the recombinant protein of the RNA-phage comprises a mutated coat protein.

In another embodiment, the core particle comprises or consists of one or more different hepatitis core (capsid) proteins (hbcags). In a related embodiment, one or more cysteine residues of these hbcags are deleted, or substituted with another amino acid residue (e.g., a serine residue). In a particular embodiment, the cysteine residues of HBcAg (corresponding to amino acid residues 48 and 107 of SEQ ID NO: 134) used to prepare the composition of the invention are deleted or substituted with another amino acid residue (e.g., a serine residue).

Furthermore, HBcAg variants used to prepare compositions of the invention are typically variants that retain the ability to bind to other hbcags, which are capable of forming dimeric or multimeric structures, presenting an ordered and repetitive antigen or antigenic determinant array.

In another embodiment, the non-natural molecular scaffold comprises or consists of: pili or pilus-like structures produced from pilin or harvested from bacteria. When pili or pilus-like structures are used to prepare the compositions of the invention, they may consist of the pilin gene naturally occurring in the bacterial cells, but modified by genetic engineering (e.g., by homologous recombination), or the product of a pilin gene introduced into these cells.

In a related embodiment, the core particle comprises or consists of: pili or pilus-like structures prepared from pilin or harvested from bacteria. These core particles may be composed of the product of a pilin gene naturally occurring in the bacterial cell.

In a particular embodiment, the formed body may comprise at least one first attachment site. The first and second attachment sites are particularly important elements of the compositions of the present invention. In various embodiments of the invention, the first and/or second attachment site may be an antigen and an antibody or antibody fragment thereof; biotin and avidin (avidin); streptavidin and biotin; a receptor and its ligand; ligand binding proteins and their ligands; an interacting leucine zipper polypeptide; an amino group and a chemical group reactive therewith; a carboxyl group and a chemical group reactive therewith; a mercapto group and a chemical group reactive thereto; or a combination thereof.

In another preferred embodiment, the composition further comprises an amino acid linker. The amino acid linker preferably comprises or consists of a second attachment site. The second attachment site mediates directed and ordered association and binding of the antigen to the core particle, respectively. An important function of the amino acid linker is to further ensure proper display and accessibility of the second attachment site, thereby facilitating binding of the antigen to the core particle, in particular by chemical cross-linking. Another important property of the amino acid linker is to further ensure optimal accessibility, in particular reactivity, of the second attachment site. These properties of the amino acid linker are more important for protein antigens.

In another preferred embodiment, the amino acid linker is selected from the group consisting of: (a) CGG; (b) an N-terminal gamma 1-linker; (c) an N-terminal gamma 3-linker; (d) an Ig hinge region; (e) an N-terminal glycine linker; (f) (G)_kC(G)_n0-12, k-0-5; (g) an N-terminal glycine-serine linker; (h) (G)_kC(G)_m(S)_l(GGGGS)_nN-0-3, k-0-5, m-0-10, l-0-2; (i) GGC; (k) GGC-NH 2; (l) A C-terminal gamma 1-linker; (m) a C-terminal γ 3-linker; (n) C-terminal GlycineA joint; (o) (G)_nC(G)_kN is 0 to 12, k is 0 to 5; (p) a C-terminal glycine-serine linker; (q) (G) _m(S)_l(GGGGS)_n(G)_oC(G)_k，n＝0-3，k＝0-5，m＝0-10，l＝0-2，o＝0-8。

An important property of glycine linkers and glycine serine linkers is their flexibility, in particular their structural flexibility, which allows for the formation of multiple conformations, which do not tend to fold into a structure that may prevent accessibility of the second attachment site. Since the glycine linker and the glycine serine linker contain no or only a limited number of side chain residues, they have a low tendency to interact extensively with the antigen, thereby further ensuring accessibility of the second attachment site. Serine residues within glycine serine linkers enhance the solubility of these linkers. Thus, the context of the present invention also includes the insertion of one or two amino acids, in particular polar or charged amino acid residues, in series or separately in a glycine or glycine serine linker.

In yet another preferred embodiment, the amino acid linker is GGC-NH2, GGC-NMe, GGC-N (Me)2, GGC-NHET, or GGC-N (Et)2, wherein the C-terminus of the cysteine residue of GGC is amidated. These amino acid linkers are preferred, particularly for embodiments in which the peptide antigen, particularly the antigen or antigenic determinant having a second attachment site, comprises an a β peptide or fragment thereof. Particularly preferred is GGC-NH 2. In another embodiment, the amino acid linker is an immunoglobulin (Ig) hinge region. Fragments of the Ig hinge region as well as Ig hinge regions modified with glycine residues are also within the scope of the present invention. Preferably, the Ig hinge region contains only one cysteine residue. It will be appreciated that this single cysteine residue of the Ig hinge region amino acid linker may be located at several positions within the linker sequence, and that the skilled person will know how to select them under the guidance of the present invention.

In one embodiment, the invention provides a method of coupling substantially all of a selected antigen to the surface of a virus, bacterial pilus, a structure formed by bacterial pilin proteins, a bacteriophage, a virus-like particle, or a viral capsid particle. In order to generate a highly effective immune response, i.e. vaccination, against the displayed antigen, the present invention exploits the strong antiviral immune response of the host by forming one antigen into a quasi-crystalline "virus-like" structure.

In another embodiment, the antigen may be selected from: (1) a protein suitable for eliciting an immune response against cancer cells; (2) proteins suitable for eliciting an immune response against infectious diseases; (3) a protein suitable for eliciting an anti-allergen immune response; (4) proteins suitable for eliciting a strong anti-autoantigen response; and (5) proteins suitable for eliciting an immune response in livestock or pets. In another embodiment, the first attachment site and/or the second attachment site is selected from: (1) genetically engineered lysine residues and (2) genetically engineered cysteine residues, which can be chemically linked together.

In yet another preferred embodiment, the first attachment site comprises or is an amino group and the second attachment site comprises or is a sulfhydryl group. Preferably, the first attachment site comprises or is a lysine residue and the second attachment site comprises or is a cysteine residue.

The invention also includes the following embodiments: the formed body particle has only one first attachment site and the antigen or antigenic determinant has only one second attachment site. Thus, when an ordered, repetitive antigen array is prepared using this embodiment, each of the formed bodies will bind to an antigen or antigenic determinant.

In another aspect, the present invention provides a composition comprising or consisting of: (a) a non-natural molecular scaffold comprising: (i) a core particle selected from a core particle of non-natural origin and a core particle of natural origin; (ii) an formed body comprising at least one first attachment site, wherein the core particle comprises or consists of: a virus-like particle, a bacterial pilus, a pilus-like structure or a modified HBcAg or fragment thereof, and the organizer is attached to the core particle by at least one covalent bond; (b) an antigen or antigenic determinant having at least one second attachment site selected from the group consisting of: (i) a non-naturally occurring attachment site for the antigen or antigenic determinant; (ii) the antigen or antigenic determinant comprises a naturally occurring attachment site, wherein the second attachment site is capable of binding to the first attachment site via at least one non-peptide bond, and the antigen or antigenic determinant interacts with the scaffold via binding to form an ordered and repetitive antigen array.

Other embodiments of the invention include methods of producing the compositions of the invention and methods of medical treatment using the vaccine compositions described herein.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.

In another aspect, the invention provides a composition comprising a bacteriophage Q β coat protein covalently linked to a phospholipase a2 protein, or fragment thereof. In a preferred embodiment, the phospholipase A enzyme₂The proteins or fragments thereof interact with bacteriophage Q β coat protein via covalent bonds to form an ordered and repetitive antigen array. In another preferred embodiment, the covalent bond is not a peptide bond. In another preferred embodiment, the phospholipase A enzyme₂The protein comprises amino acids selected from the group consisting of: SEQ ID NO: 168, the amino acid sequence of SEQ ID NO: 169, seq id NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, the amino acid sequence of SEQ ID NO: 173, SEQ ID NO: 174 and SEQ ID NO: 175.

The invention also provides a method for producing the composition, comprising contacting bacteriophage Q beta coat protein with phospholipase A₂Protein combinations in which bacteriophage Q beta coat protein is associated with phospholipase A₂The proteins interact to form an antigen array.

In another aspect, the invention also provides a composition comprising a non-native molecular scaffold comprising bacteriophage Q β coat protein, and a recombinant host cell comprising the non-native molecular scaffoldAn organiser comprising at least one first attachment site, wherein the organiser is linked to bacteriophage Q β coat protein by at least one covalent bond; and phospholipase A having at least a second attachment site₂A protein or fragment thereof or variant thereof, the second attachment site being selected from the group consisting of: phospholipase A₂A non-naturally occurring attachment site for a protein or fragment thereof; phospholipase A₂A naturally occurring attachment site for a protein or fragment thereof, wherein the second attachment site is bound to the first attachment site by at least one non-peptide bond, and the antigen or antigenic determinant interacts with the scaffold by such binding to form an ordered and repetitive antigen array. In a preferred embodiment, the phospholipase A enzyme₂The protein comprises amino acids selected from the group consisting of: SEQ ID NO: 168, the amino acid sequence of SEQ ID NO: 169, the amino acid sequence of SEQ ID NO: 170, seq id NO: 171, SEQ ID NO: 172, the amino acid sequence of SEQ ID NO: 173, SEQ ID NO: 174 and SEQ ID NO: 175.

The invention also provides a method for producing the composition, comprising contacting bacteriophage Q beta coat protein with phospholipase A₂Protein combinations in which bacteriophage Q beta coat protein is associated with phospholipase A₂The proteins interact to form an antigen array. The antigen array is preferably ordered and/or repetitive.

The invention also provides a pharmaceutical composition comprising phospholipase A₂Protein and a pharmaceutically acceptable carrier. The invention also provides a composition comprising phospholipase A₂Vaccine compositions of proteins. In a preferred embodiment, the vaccine composition of claim 31 further comprises at least one adjuvant.

The present invention also provides a method for treating allergy caused by bee venom, comprising administering the pharmaceutical composition or vaccine composition to a patient. As a result of this administration, patients show a reduced immune response to bee venom.

The invention also relates to a vaccine for preventing prion-mediated diseases by inducing anti-lymphotoxin beta, anti-lymphotoxin alpha, or anti-lymphotoxin beta receptor antibodies. The vaccine comprises a protein carrier foreign to the human or animal to be immunized, which is coupled to lymphotoxin beta or a fragment thereof, lymphotoxin alpha or a fragment thereof, or lymphotoxin beta receptor or a fragment thereof. The vaccine is injected into a human or animal in order to induce antibodies specific for endogenous lymphotoxin beta, lymphotoxin alpha or lymphotoxin beta receptors. The induced anti-lymphotoxin beta, lymphotoxin alpha, or anti-lymphotoxin beta receptor antibody reduces or eliminates the follicular dendritic cell pool present in lymphoid organs. Since prion replication in lymphoid organs and transport to the central nervous system is affected in the absence of follicular dendritic cells, this treatment inhibits the development of prion-mediated diseases. In addition, blocking lymphotoxin beta is beneficial to autoimmune disease (e.g., type I diabetes) patients.

Brief Description of Drawings

FIGS. 1A-1C: a modular eukaryotic expression vector for expressing the antigen of the invention;

FIGS. 2A-2C: cloning, expression and coupling of resistin to Q β capsid protein;

FIGS. 3A-3B: for conjugation to virus-like particles and pili, cloning and expression of lymphotoxin-beta constructs.

FIGS. 4A-4B: cloning, expression and coupling of MIF constructs to Q β capsid proteins.

FIG. 4C: ELISA analysis of MIF-specific IgG antibodies in sera of mice immunized with MIF protein coupled to Q β capsid protein.

FIG. 5: coupling of MIF constructs to fr capsid protein and to HBcAg-lys-2Cys-Mut capsid protein as analyzed by SDS-PAGE.

FIG. 6: cloning and expression of human C-RANKL.

FIG. 7: cloning and expression of prion protein.

FIG. 8A: ELISA analysis of "Angio I" specific IgG antibodies in sera of mice immunized with angiotensin conjugated to Q β capsid protein.

FIG. 8B: ELISA analysis of "Angio II" specific IgG antibodies in sera of mice immunized with angiotensin conjugated to Q β capsid protein.

FIG. 8C: ELISA analysis of "Angio III" specific IgG antibodies in sera of mice immunized with angiotensin conjugated to Q β capsid protein.

FIG. 8D: ELISA analysis of "Angio IV" specific IgG antibodies in sera of mice immunized with angiotensin conjugated to Q β capsid protein.

FIG. 9A: ELISA analysis of "Der pI p 52" specific IgG antibodies in sera of mice immunized with Der pI peptide coupled to Q β capsid protein.

FIG. 9B: ELISA analysis of "Der pI p 117" specific IgG antibodies in sera of mice immunized with Der pI peptide coupled to Q β capsid protein.

FIG. 10A: ELISA analysis of IgG antibodies specific for human VEGFR II peptide in mouse sera immunized with human VEGFR II peptide coupled to type I pilin and human VEGFR II ectodomain.

FIG. 10B: ELISA analysis of IgG antibodies specific for the ectodomain of human VEGFR II in mouse sera immunized with human VEGFR II peptide coupled to type I pilin and human VEGFR II ectodomain.

FIG. 11: ELISA analysis of anti-TNF α protein-specific IgG antibodies in sera of mice immunized with full-length HBc-TNF.

FIG. 12: ELISA analysis of anti-TNF α protein-specific IgG antibodies in sera of mice immunized with 2cysLys-mut HBcAg1-149 conjugated to 3' TNF II peptide.

FIG. 13A: "A β 1-15" was analyzed by SDS-PAGE coupled to Q β capsid protein using the cross-linking agent SMPH.

FIG. 13B: "A β 33-42" was analyzed by SDS-PAGE coupled to Q β capsid protein using the cross-linking agent SMPH.

FIG. 13C: "A β 1-27" was analyzed by SDS-PAGE coupled to Q β capsid protein using the cross-linking agent SMPH.

FIG. 13D: "A β 1-15" was analyzed by SDS-PAGE with Q β capsid protein coupled with the crosslinker Sulfo-GMBS.

FIG. 13E: "A β 1-15" was analyzed by SDS-PAGE coupled to Q β capsid protein using the crosslinker Sulfo-MBS.

FIG. 14A: ELISA analysis of "Abeta 1-15" specific IgG antibodies in sera of mice immunized with "Abeta 1-15" coupled to Q beta capsid protein.

FIG. 14B: ELISA analysis of "Abeta 1-27" specific IgG antibodies in sera of mice immunized with "Abeta 1-27" coupled to Q beta capsid protein.

FIG. 14C: ELISA analysis of "Abeta 33-42" specific IgG antibodies in sera of mice immunized with "Abeta 33-42" coupled to Q beta capsid protein.

FIG. 15A: SDS-PAGE analysis of pCC2 coupled to Q β capsid protein.

FIG. 15B: SDS-PAGE analysis of pCA2 coupled to Q β capsid protein.

FIG. 15C: SDS-PAGE analysis of coupling of pCB2 to Q β capsid protein.

FIG. 16: coupling of prion peptides to Q β capsid protein; SDS-PAGE analysis.

FIG. 17A: SDS-PAGE analysis of IL-5 expression in bacteria.

FIG. 17B: Western-Blot analysis of IL-5 and IL-13 expression in eukaryotic cells.

FIG. 18A: SDS-PAGE analysis of murine VEGFR-2 peptide coupled to pili.

FIG. 18B: SDS-PAGE analysis of murine VEGFR-2 peptide coupled to Q β capsid protein.

FIG. 18C: SDS-PAGE analysis of murine VEGFR-2 peptide coupled to HBcAg-lys-2 cys-Mut.

FIG. 18D: ELISA analysis of IgG antibodies specific for murine VEGFR-2 peptide in sera from mice immunized with murine VEGFR-2 peptide coupled to pili.

FIG. 18E: ELISA analysis of IgG antibodies specific for murine VEGFR-2 peptide in sera from mice immunized with murine VEGFR-2 peptide conjugated to Q β capsid protein.

FIG. 18F: ELISA analysis of IgG antibodies specific for murine VEGFR-2 peptide in sera from mice immunized with murine VEGFR-2 peptide coupled to HBcAg-lys-2 cys-Mut.

FIG. 19A: SDS-PAGE analysis of Abeta 1-15 peptide coupled to HBcAg-lys-2cys-Mut and fr capsid proteins.

FIG. 19B: ELISA analysis of IgG antibodies specific for A.beta.1-15 in sera of mice immunized with A.beta.1-15 peptide conjugated to HBcAg-lys-2cys-Mut or fr capsid protein.

FIG. 20: ELISA analysis of human Α β -specific IgG antibodies in sera of transgenic APP23 mice immunized with human Α β peptide coupled to Q β capsid protein.

FIG. 21: SDS-PAGE analysis of Fab antibody fragments coupled to Q.beta.capsid protein.

FIG. 22A: the flag peptide was analyzed by SDS-PAGE with the crosslinker sulfo GMBS coupled to the mutated Q.beta.capsid protein.

FIG. 22B: the flag peptide was analyzed by SDS-PAGE with the coupling of the cross-linker sulfo MBS to the mutant Q.beta.capsid protein.

FIG. 22C: the flag peptide was analyzed by SDS-PAGE with the coupling of the mutant Q.beta.capsid protein by the cross-linking agent SMPH.

FIG. 22D: PLA (polylactic acid)₂Coupling of the cys protein with the mutant Q.beta.capsid protein by means of the crosslinking agent SMPHSDS-PAGE analysis of (5).

FIG. 23: ELISA assay using M2 peptide immunization coupled to mutant Q β capsid protein and fr capsid.

FIG. 24: DER p1, SDS-PAGE analysis of peptide 2 coupled to mutant Q β capsid proteins.

FIG. 25A: desensitization of allergic mice with PLA2 coupled to Q β capsid protein: and (6) measuring the temperature.

FIG. 25B: desensitization of allergic mice with PLA2-cys coupled to Q β capsid protein: IgG 2A and Ig E titers.

FIG. 26: PLA (polylactic acid)₂SDS-PAGE analysis and Western-blot analysis of cys coupled to Q.beta.capsid proteins.

Fig. 27A: ELISA analysis of IgG antibodies specific for the M2 peptide in sera of mice immunized with the M2 peptide coupled to HBcAg-lys-2cys-Mut, Q β capsid protein, fr capsid protein, HBcAg-lys-1-183 and the M2 peptide fused to HBcAg 1-183.

Fig. 28A: SDS-PAGE analysis of anti-idiotype IgE mimobody VAE051 coupled to Q β capsid proteins.

FIG. 28B: ELISA analysis of anti-idiotypic antibodies VAE051 and human IgE-specific Ig antibodies in sera of mice immunized with VAE051 coupled to Q β capsid protein.

Detailed Description

1. Definition of

A virus: as used herein, the term "alphavirus" refers to all RNA viruses encompassed by the genus alphavirus. Strauss and Strauss, microbiol.rev., 58: 491, 562(1994) describes members of this genus. Examples of alphaviruses include: orlao virus, Bibaru virus, armadillo virus, chikungunya virus, eastern equine encephalitis virus, Morgan castle virus, Gatta virus, Cruzziqi virus, Malaria virus, Midelburg virus, Mucanbo virus, Endurum virus, Pickerona virus, Tornatt virus, Trinidi virus, Urna virus, Western equine encephalitis virus, Wadarlo river virus, Sindbis virus (SIN), Semliki Forest Virus (SFV), Venezuelan equine encephalitis Virus (VEE), and Ross river virus.

Antigen: as used herein, the term "antigen" is a molecule capable of being bound by an antibody. Antigens can also induce a humoral and/or cellular immune response, leading to the production of B-and/or T-lymphocytes. The antigen may contain one or more epitopes (B-epitope and T-epitope). What is meant by specific reaction is that an antigen will react in a highly selective manner with its corresponding antibody, but not with most other antibodies induced by other antigens.

Antigenic determinant: as used herein, the term "antigenic determinant" refers to the portion of an antigen that is specifically recognized by B-lymphocytes or T-lymphocytes. B-lymphocytes react to foreign antigenic determinants by producing antibodies, while T-lymphocytes are mediators of cellular immunity. Thus, an antigenic determinant or epitope is the portion of an antigen that is recognized by an antibody, or in the case of MHC, by a T cell receptor.

Combining: as used herein, the term "bind" when used in reference to first and second attachment sites means at least one non-peptide bond. The nature of the binding may be covalent, ionic, hydrophobic, polar or any combination thereof.

First attachment site: as used herein, the phrase "first attachment site" refers to an element of the "formed body" which itself binds to the core particle in a non-random manner to which a second attachment site located on an antigen or antigenic determinant may bind. The first attachment site can be a protein, polypeptide, amino acid, peptide, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ion, phenylmethylsulfonyl fluoride), or a combination thereof, or a chemically reactive group thereof. In the repeating configuration, there are multiple first attachment sites on the surface of the non-native molecular scaffold.

A second attachment site: as used herein, the phrase "second attachment site" refers to an element associated with an antigen or antigenic determinant to which the first attachment site of a "formed body" located on the surface of a non-natural molecular scaffold can bind. The second attachment site for an antigen or antigenic determinant may be a protein, polypeptide, peptide, sugar, polynucleotide, natural or synthetic polymer, secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ion, phenylmethylsulfonyl fluoride), or a combination thereof, or a chemically reactive group thereof. At least one second attachment site is present on the antigen or antigenic determinant. Thus, the term "antigen or antigenic determinant comprising at least one second attachment site" refers to an antigen or antigenic structure comprising at least an antigen or antigenic determinant and a second attachment site. However, especially for second attachment sites that do not naturally occur within antigens or antigenic determinants, these antigens or antigenic structures comprise an "amino acid linker". Such an amino acid linker, or "linker" as used herein, may bind the antigen or antigenic determinant and the second attachment site, or more preferably, already comprise or contain a second attachment site, typically, but not necessarily, an amino acid residue, preferably a cysteine residue. However, the term "amino acid linker" as used herein does not mean that such an amino acid linker is composed of only amino acid residues, although an amino acid linker composed of amino acid residues is a preferred embodiment of the present invention. The amino acid residues in the amino acid linker preferably consist of naturally occurring amino acids or non-natural amino acids, both in the L-form or both in the D-form or mixtures thereof, as is well known in the art. However, the invention also includes amino acid linkers comprising molecules with sulfhydryl or cysteine residues. Such molecules preferably comprise C1-C6 alkyl-, cycloalkyl (C5, C6), aryl or heteroaryl moieties. The antigen or antigenic determinant or optionally the second attachment site is preferably bound to the amino acid linker by at least one covalent bond, more preferably by at least one peptide bond.

In combination: as used herein, the term "associated" refers to binding or attachment, which may be covalent, such as chemical coupling, or non-covalent, such as ionic interactions, hydrophobic interactions, hydrogen bonding, and the like. The covalent bond may be, for example: esters, ethers, phosphoesters, amides, peptides, imides, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. The term "conjugated" is broader than, and includes, terms such as "coupled", "fused", and "attached".

Core particles: as used herein, the term "core particle" refers to a rigid structure with an inherent repeating organization that provides the basis for the attachment of "formers". The core particle as used herein may be the product of a synthetic process or the product of a biological process.

Coat protein: as used herein, the term "coat protein" refers to a protein of a bacteriophage or RNA-bacteriophage that is capable of participating in phage or RNA-bacteriophage capsid assembly. However, the term "CP" is used when referring to a specific gene product of an RNA-phage coat protein gene. For example, the specific gene product of the coat protein gene of PNA-phage Q β is referred to as "Q β CP", while the "coat protein" of phage Q β includes "Q β CP" as well as the A1 protein.

Cis-acting: as used herein, the term "cis-acting" sequence refers to a nucleic acid sequence to which a replicase binds thereby catalyzing RNA-dependent replication of an RNA molecule. These replication events result in the replication of both full-length and partial RNA molecules, and thus, the alphavirus subgenomic promoter is also a "cis-acting" sequence. The cis-acting sequence may be located at or near the 5 'end, 3' end, or both ends of the nucleic acid molecule, as well as internal.

Fusing: as used herein, the term "fusion" refers to the combination of amino acid sequences of different origin in one polypeptide chain by in-frame combination of their encoding nucleotide sequences. The term "fusion" obviously also includes, in addition to fusion to one of its ends, internal fusion, i.e. insertion of sequences of different origin into a polypeptide chain.

Heterologous sequence: as used herein, the term "heterologous sequence" refers to a second nucleotide sequence present in a vector of the invention. The term "heterologous sequence" also refers to any amino acid or RNA sequence encoded by the heterologous DNA sequence comprised in the vector of the invention. The heterologous nucleotide sequence can encode a protein or RNA molecule that is normally expressed in the cell type containing the sequence, or a molecule that is not normally expressed therein (e.g., a Sindbis structural protein).

Separating: as used herein, the term "isolated" when applied to a molecule means that the molecule has been removed from its natural environment. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from coexisting materials in its natural state is "isolated. Furthermore, for the purposes of the present invention, a recombinant DNA molecule contained in a vector is considered to be isolated. Isolated RNA molecules include in vivo or in vitro RNA replication products of DNA and RNA molecules. Isolated nucleic acid molecules also include synthetically produced molecules. In addition, the vector molecule contained in the recombinant host cell is also isolated. Thus, not all "isolated" molecules need to be "purified".

Immunotherapeutic agents: as used herein, the term "immunotherapeutic agent" is a composition for the treatment of a disease or disorder. More specifically, the term is used to refer to a method of treatment of allergy or a method of treatment of cancer.

Individual: as used herein, the term "individual" refers to a multicellular organism, including plants and animals. Preferred multicellular organisms are animals, more preferably vertebrates, even more preferably mammals, and most preferably humans.

Low or undetectable: as used herein, the phrase "low or undetectable" when used in reference to a gene expression level means that the expression level is significantly below (e.g., at least 5-fold lower than) that seen when the gene is maximally induced, or is not readily detectable by the methods used in the examples section below.

Lectin: as used herein, refers to proteins obtained from seeds of inter alia leguminous plants, as well as from a variety of other plant and animal sources, which contain binding sites for specific mono-or oligosaccharides. Examples include concanavalin a and wheat germ agglutinin, which are widely used as reagents for analysis and preparation in glycoprotein studies.

Analog bit: as used herein, the term "mimotope" refers to a substance that elicits an immune response to an antigen or antigenic determinant. The term mimotope is generally used for a particular antigen. For example, stimulation of anti-phospholipase A₂(PLA₂) The peptide produced by the antibody is a mimotope of the antigenic determinant to which the antibody binds. The mimotopes may or may not have substantial structural similarity, or share or not share structural features, with the antigen or antigenic determinant against which the immune response is elicited. Methods for generating and identifying mimotopes that elicit an immune response against a particular antigen or antigenic determinant are well known in the art and are described elsewhere herein.

The natural sources are as follows: as used herein, the term "naturally-derived" means that none, in whole or in part, is synthetic, but naturally-occurring or produced.

Non-natural: as used herein, the term generally refers to not being from nature, and more specifically, the term refers to being from man-made.

Non-natural sources: as used herein, the term "non-natural source" generally refers to a source that is synthetic or not from nature, and more specifically, the term refers to a source that is artificial.

Non-natural molecular scaffolds: as used herein, the phrase "non-natural molecular scaffold" refers to any product that is artificially prepared and can be used to create a rigid, repeating array of first attachment sites. Ideally, but not necessarily, these first attachment sites have a geometric order. The non-natural molecular scaffold may be organic or inorganic in part or in whole, and may be chemically synthesized or synthesized by biological methods. The composition of the non-natural molecular scaffold is as follows: (a) a core particle of natural or unnatural origin; and (b) an formed body which itself comprises at least one first attachment site and is linked to the core particle by at least one covalent bond. In a particular embodiment, the non-natural molecular scaffold can be a virus, a virus-like particle, a bacterial pilus, a viral capsid particle, a bacteriophage, a recombinant form thereof, or a synthetic particle.

Ordered, repetitive antigen or antigenic determinant array: as used herein, the term "ordered, repetitive antigen or antigenic determinant array" generally refers to a repetitive pattern of antigens or antigenic determinants characterized by a uniform spatial arrangement of antigens or antigenic determinants relative to a non-natural molecular scaffold. In one embodiment of the invention, the repeating pattern may be a geometric pattern. Examples of suitable ordered, repetitive antigen or antigenic determinant arrays are those having paracrystalline sequences in which the antigens or antigenic determinants repeat strictly at 5-15 nanometer intervals.

Forming a body: as used herein, the term "formed body" refers to an element that is bound to a core particle in a non-random manner, providing nucleation sites for the production of an ordered and repetitive antigen array. An organizer is any element comprising at least one first attachment site that is bound to the core particle by at least one covalent bond. The formers may be proteins, polypeptides, peptides, amino acids (i.e., residues of proteins, polypeptides, or peptides), sugars, polynucleotides, natural or synthetic polymers, secondary metabolites or compounds (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl fluoride), or combinations thereof, or chemically reactive groups thereof. Thus, the formed bodies further ensure the formation of an ordered, repetitive antigen array of the present invention. In typical embodiments of the invention, the core particle is modified, e.g., by genetic engineering or chemical reaction, to produce a non-natural molecular scaffold comprising the core particle and a former, wherein the former is attached to the core particle by at least one covalent bond. In certain embodiments of the invention, however, the selected formed body is part of the core particle. Thus, for these embodiments, the creation of a non-natural molecular scaffold comprising a core particle and an organiser and the ensuring that an ordered, repetitive antigen array is formed do not necessarily require modification of the core particle.

The allowable temperature: as used herein, the phrase "permissive temperature" refers to a temperature at which the enzyme has a relatively high level of catalytic activity.

And (3) pilus: as used herein, the term "pilus" (pilus in the singular) refers to the extracellular structure of bacterial cells composed of protein monomers (e.g., pilin monomers) organized in an ordered, repeating pattern. Moreover, pili are structures that participate in the following processes: such as bacterial cell attachment to host cell surface receptors, intercellular gene exchange, and cell-cell recognition. Examples of pili include type I pili, P-pili, F1C pili, S-pili, and 987P-pili. Other examples of pili are described below.

Pilus-like structure: as used herein, the phrase "pilus-like structure" refers to a structure that has characteristics similar to pili and is composed of protein monomers. An example of a "pilus-like structure" is a structure formed by bacterial cells expressing modified pilin proteins that do not form an ordered, repeating array that is substantially identical to native pili.

Polypeptide: as used herein, the term "polypeptide" refers to a polymer composed of amino acid residues, usually natural amino acid residues, joined together by peptide bonds. Although the polypeptide need not be limited in size, the term polypeptide is generally used for peptides of about 10-50 amino acids in size.

Protein: as used herein, the term protein refers to polypeptides of generally more than 20, especially more than 50 amino acid residues in size. Proteins generally have a defined three-dimensional structure, although they do not necessarily have to be, and are often referred to as folded, and instead, peptides and polypeptides that do not generally have a defined three-dimensional structure, but rather adopt a large number of different conformations, are referred to as unfolded. The defined three-dimensional structure of the protein is particularly important for the binding between the core particle and the antigen, which is mediated by the second attachment site, in particular by chemical cross-linking between the first and second attachment sites using a chemical cross-linking agent. In certain aspects of the invention, the amino acid linker is also closely related to the structural characteristics of the protein.

Purification of: as used herein, the term "purified" when used with respect to a molecule means that the molecule is purified at an increased concentration relative to the molecule with which it is associated in its natural environment. Naturally associated molecules include proteins, nucleic acids, lipids, and sugars, but generally do not include water, buffers, and reagents added to maintain integrity or facilitate purification of the molecule. For example, in oligo dT column chromatography, mRNA molecules are purified by such chromatography even if the naturally associated nucleic acids and other biomolecules are not bound to the column and separated from the mRNA molecule of interest, even if the mRNA is diluted with an aqueous solvent.

Receptor: as used herein, the term "receptor" refers to a protein or glycoprotein or fragment thereof that is capable of interacting with another molecule, known as a ligand. The ligand may belong to any class of biochemical or chemical compounds. The receptor does not necessarily have to be a membrane-bound protein. Soluble proteins, such as maltose binding protein or retinol binding protein, are also receptors.

Residue: as used herein, the term "residue" means a particular amino acid in a polypeptide backbone or side chain.

Recombinant host cell: as used herein, the term "recombinant host cell" refers to a host cell into which one or more of the nucleic acid molecules of the invention has been introduced.

Recombinant virus: as used herein, the phrase "recombinant virus" refers to an artificially genetically modified virus. This phrase includes all viruses known in the art. More specifically, the phrase refers to an alphavirus that is artificially genetically modified, and most specifically, the phrase refers to an sindbis virus that is artificially genetically modified.

Limiting temperature: as used herein, the phrase "limiting temperature" refers to a temperature at which an enzyme has a low level or undetectable catalytic activity. Both "hot" and "cold" sensitive mutants are well known. Thus, the limiting temperature may be higher or lower than the allowable temperature.

RNA-dependent RNA replication events: as used herein, the phrase "RNA-dependent RNA replication event" refers to a process that results in the formation of an RNA molecule using an RNA molecule as a template.

RNA-dependent RNA polymerase: as used herein, the phrase "RNA-dependent RNA polymerase" refers to a polymerase that catalyzes the production of one RNA molecule from another RNA molecule. The term is synonymous herein with the term "replicase".

RNA-phage: as used herein, the term "RNA-phage" refers to a bacterium-infecting RNA virus, preferably a bacterium-infecting single-stranded positive-sense RNA virus.

Self-antigen: as used herein, the term "autoantigen" refers to a protein encoded by the DNA of a host, the product produced by the protein or RNA encoded by the DNA of the host being defined as self. In addition, proteins produced by the combination of two or more self-molecules or proteins that are part of self-molecules, as well as proteins with high homology (> 95%) to the above self-molecules, can also be considered to be self-derived.

Temperature-sensitive: as used herein, the phrase "temperature sensitive" refers to an enzyme that readily catalyzes a reaction at one temperature, but catalyzes the same reaction more slowly or not at all at another temperature. An example of a temperature sensitive enzyme is the replicase protein encoded by the pCYTts vector, which has readily detectable replicase activity at temperatures below 34 ℃ and less or undetectable at 37 ℃.

Transcription: as used herein, the term "transcription" refers to the production of an RNA molecule from a DNA template catalyzed by an RNA polymerase.

Non-translated RNA: as used herein, the phrase "untranslated RNA" refers to an RNA sequence or molecule that does not encode an open reading frame, or encodes an open reading frame or portion thereof, but is in a form that does not produce an amino acid sequence (e.g., does not contain a start codon). Examples of such molecules are tRNA molecules, rRNA molecules and ribozymes.

Carrier: as used herein, the term "vector" refers to an agent (e.g., a plasmid or virus) used to transfer genetic material into a host cell. The vector may consist of DNA or RNA.

Virus-like particles: as used herein, the term "virus-like particle" refers to a structure that is similar to a viral particle. Furthermore, the virus-like particle of the present invention is non-replicative and non-infectious because it lacks all or part of the viral genome, in particular the replicative and infectious components of the viral genome. The virus-like particle of the present invention may contain nucleic acids other than its genome.

Virus-like particles of bacteriophage: as used herein, the term "bacteriophage virus-like particle" refers to a virus-like particle similar to the structure of a bacteriophage, non-replicating and non-infectious, lacking at least the genes encoding the replication machinery of the bacteriophage, and generally also the genes encoding the proteins responsible for viral attachment or entry into the host. However, this definition also includes such phage virus-like particles, wherein the above genes are still present but inactivated, thus also producing non-replicative and non-infectious phage virus-like particles.

Viral particles: the term "viral particle" as used herein refers to the morphological form of a virus. In certain virus types, the viral particle comprises a genome surrounded by a protein capsid; others have additional structure (e.g., capsule, tail, etc.).

One of them: when the terms "a," "an," and "an" are used in this disclosure, they mean "at least one" or "one or more" unless otherwise indicated.

2. Ordered and repetitive antigen or antigenic determinant array compositions and methods for making same

The present invention provides compositions comprising ordered and repetitive antigens or antigenic determinant arrays. In addition, the present invention enables the skilled artisan to conveniently construct ordered and repetitive antigen or antigenic determinant arrays for various therapeutic purposes, including the prevention of infectious diseases, the treatment of allergies, and the treatment of cancer.

The composition of the invention essentially comprises or consists of the following two elements: (1) a non-natural molecular scaffold; and (2) an antigen or antigenic determinant comprising at least one second attachment site capable of binding to the first attachment site via at least one non-peptide bond.

The composition of the invention also comprises or consists of: bacterial pilin proteins directly linked to an antigen or antigenic determinant.

This non-natural molecular scaffold comprises or consists of the following elements: (a) a core particle selected from: (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (b) an formed body comprising at least one first attachment site, wherein the formed body is linked to the core particle by at least one covalent bond.

The composition of the invention also comprises or consists of: a core particle to which an antigen or antigenic determinant is directly linked.

The antigen or antigenic determinant comprises at least one second attachment site selected from the group consisting of: (a) a non-naturally occurring attachment site for the antigen or antigenic determinant; (b) the naturally occurring attachment site for the antigen or antigenic determinant.

The present invention provides an ordered and repetitive antigen array by binding the second attachment site to the first attachment site via at least one non-peptide bond. Thus, by this binding of the first attachment site to the second attachment site, the antigen or antigenic determinant is bound to the unnatural molecular scaffold to form an ordered and repetitive antigen array.

The skilled artisan can specifically design the antigen or antigenic determinant and the second attachment site such that the arrangement of all antigens or antigenic determinants bound to the non-natural molecular scaffold-or in certain embodiments to the core particle-is uniform. For example, a single second attachment site may be placed at the carboxy-or amino-terminus of an antigen or antigenic determinant, thereby ensuring by design that all antigen or antigenic determinant molecules attached to the non-natural molecular scaffold are located in a uniform manner. Thus, the present invention provides a convenient way to place any antigen or antigenic determinant in a defined order on a non-natural molecular scaffold in a manner that forms a repeating pattern.

It will be clear to those skilled in the art that certain embodiments of the present invention relate to the use of recombinant nucleic acid techniques such as cloning, polymerase chain reaction, purification of DNA and RNA, expression of recombinant proteins in prokaryotic and eukaryotic cells, and the like. Such methods are well known to those skilled in the art and are readily found in published laboratory methods manuals (e.g., molecular cloning: A laboratory Manual, 2 nd edition, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y. (1989); modern methods of molecular biology, John H.Wiley & Sons, Inc. (1997), by Sambrook, J. et al). The basic Laboratory techniques for studies using tissue culture cell lines (Celis, cell biology, Academic Press, 2 nd edition, (1998) written by J.) and antibody-based techniques (Harlow, E. and Lane, D., "antibodies: A Laboratory Manual"; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988); Deutscher, M.P., "protein purification guide"; methods in enzymology, 128, Academic Press Diego (1990); Scopes, R.K., "principles and practices for protein purification", 3 rd edition, Springer-Verlag, New York (1994)) are also well described in the literature, and are all incorporated herein by reference.

A. Core particles and non-natural molecular scaffolds

One component of certain compositions of the invention is a non-natural molecular scaffold comprising or consisting of a core particle and a former. As used herein, the phrase "non-natural molecular scaffold" refers to any artificially produced product that can be used to provide a rigid, repeating array of first attachment sites. More specifically, the non-natural molecular scaffold comprises or consists of the following elements: (a) a core particle selected from: (1) a core particle of non-natural origin and (2) a core particle of natural origin; and (b) an formed body comprising at least one first attachment site, wherein the formed body is linked to the core particle by at least one covalent bond.

One skilled in the art will readily recognize that the core particle of the non-natural molecular scaffold of the present invention is not limited to any particular form. The core particles may be organic or inorganic, and may be chemically synthesized or synthesized by biological methods.

In one embodiment, the non-natural core particle may be a synthetic polymer, lipid micelle, or metal. Such core particles are well known in the art and provide the basis for the establishment of the novel non-natural molecular scaffold of the present invention. For example, U.S. patent No. 5,770,380 describes synthetic polymer or metal core particles, disclosing the use of calixarene organic scaffolds linked to multiple peptide loops for the production of "mimetibodies," and U.S. patent No. 5,334,394 describes nanocrystalline particles for use as viral baits, composed of a variety of inorganic materials, including metals or ceramics. Suitable metals include chromium, rubidium, iron, zinc, selenium, nickel, gold, silver, platinum. Suitable ceramic materials in this embodiment include silica, titania, alumina, ruthenium oxide, and tin oxide. The core particles of this embodiment may be made of organic matter including carbon (diamond). Suitable polymers include polystyrene, nylon, and nitrocellulose. For this type of nanocrystalline particles, particles made of tin oxide, titanium dioxide, or carbon (diamond) may also be used. Lipid micelles may be prepared by any method known in the art. For example, micelles can be prepared by the following method: baisell and Millar (Biophys. chem.4: 355-361(1975)) or Corti et al (chem. Phys. lipids 38: 197-214(1981)) or Lopez et al (FEBS Lett.426: 314-318(1998)) or Topchieva and Karezin (J. Colloid Interface Sci.213: 29-35(1999)) or Morein et al (Nature 308: 457-460(1984)), both of which are incorporated herein by reference.

The core particle may also be produced by biological methods, and may be natural or non-natural. For example, such embodiments may include a core particle comprising or alternatively consisting of a virus, virus-like particle, bacterial pilus, bacteriophage, viral capsid particle, or recombinant form thereof. In a more specific embodiment, the core particle may comprise or consist of: recombinant proteins of rotavirus, norwalk virus, alphavirus, bacterial pilus or pilus-like structures, foot and mouth disease virus, retrovirus, hepatitis B virus (such as HBcAg), tobacco mosaic virus, aviary hut virus and human papilloma virus. The core particle may also comprise or consist of: one or more fragments of these proteins, and variants of these proteins, that retain the ability to bind to each other, form an ordered and repetitive antigen or antigenic determinant array.

As described in more detail below, protein variants that retain the ability to bind to each other, form an ordered, repetitive antigen or antigenic determinant array, may be, for example, at least 80%, 85%, 90%, 95%, 97%, or 99% identical at the amino acid level to their wild-type counterparts. The method comprises the following steps of: 89, the invention includes vaccine compositions comprising an HBcAg polypeptide comprising or consisting of: and SEQ ID NO: 89, and an amino acid sequence which is at least 80%, 85%, 90%, 95%, 97% or 99% identical to the amino acid sequence shown in 89, and, if desired, a protein form which has been processed to remove the N-terminal leader sequence. These variants are typically capable of binding to form dimeric or multimeric structures. Methods that can be used to determine whether a protein forms such a structure include gel filtration, agarose gel electrophoresis, sucrose gradient centrifugation, and electron microscopy (e.g., Koschel, M. et al, J.Virol 73: 2153-.

Protein fragments that retain the ability to bind to each other, form an ordered, repetitive antigen or antigenic determinant array may comprise or consist of the following polypeptides: a polypeptide 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids in length. Examples of such protein fragments include those described herein as being suitable for preparing core particles and/or non-native molecular scaffolds.

Whether natural or non-natural, the core particles of the present invention generally have a former that is attached to the natural or non-natural core particle by at least one of its valences. The former is an element that binds to the core particle in a non-random manner, providing a nucleation site for the generation of an ordered, repetitive antigen array. Desirably, but not necessarily, the former is combined with the core particle in geometric order. The formation comprises at least one first attachment site.

In some embodiments of the invention, an ordered and repetitive array is formed by the association between (1) a core particle or non-natural molecular scaffold and (2) (a) one antigen or antigenic determinant or (b) one or more antigens or antigenic determinants. For example, bacterial pili or pilus-like structures are composed of proteins organized into ordered, repeating structures. Thus, in many cases, an ordered array of antigens or antigenic determinants can be formed by linking these components directly to a bacterial pilus or pilus-like structure or by a organiser.

As previously mentioned, the formed body may be any element comprising at least one first attachment site that is bound to the core particle by at least one covalent bond. The formers may be proteins, polypeptides, peptides, amino acids (i.e., residues of proteins, polypeptides, or peptides), sugars, polynucleotides, natural or synthetic polymers, secondary metabolites or compounds (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonyl fluoride), or combinations thereof, or chemically reactive groups thereof. In a more specific embodiment, the formed body may comprise a first attachment site comprising: an antigen, an antibody or antibody fragment, biotin, avidin, streptavidin, a receptor ligand, a ligand binding protein, an interacting leucine zipper polypeptide, an amino group, a chemical group reactive with an amino group, a carboxyl group, a chemical group reactive with a carboxyl group, a sulfhydryl group, a chemical group reactive with a sulfhydryl group, or a combination thereof.

In one embodiment, the core particle of the non-natural molecular scaffold comprises a virus, bacterial pilus, a structure formed from bacterial pilus proteins, a bacteriophage, a virus-like particle, a viral capsid particle, or a recombinant form thereof. Any virus known in the art having an ordered and repetitive structure of the shell and/or core proteins may be selected as the non-native molecular scaffold of the present invention. Examples of suitable viruses include: sindbis virus and other alphaviruses, rhabdoviruses (e.g., vesicular stomatitis virus), picornaviruses (e.g., human rhinovirus, Aichi virus), togaviruses (e.g., rubella virus), orthomyxoviruses (e.g., sogato virus, fibonan virus, fowl plague virus), polyomaviruses (e.g., polyomavirus BK, polyomavirus JC, bird polyomavirus BFDV), parvoviruses, rotaviruses, phage Q β, phage R17, phage M11, phage MX1, phage NL95, phage fr, phage GA, phage SP, phage MS2, phage f2, phage PP7, norwalk virus, foot and mouth disease virus, retrovirus, hepatitis b virus, tobacco mosaic virus, aviary hut virus, and human papillomavirus (see, for example, Bachman, m.f. and Zinkernagel, r.m., unal. tdayy virus (558).

In one embodiment, the invention utilizes genetic engineering of viruses to produce fusions of ordered and repetitive viral envelope proteins with formed bodies comprising selected heterologous proteins, peptides, antigenic determinants, or reactive amino acid residues. Other genetic manipulations well known to those skilled in the art may be included in the construction of the non-native molecular scaffold; for example, it may be desirable to limit the replication capacity of a recombinant virus by genetic mutation. The viral protein selected for fusion with the formative (i.e., first attachment site) protein should have an organized, repetitive structure. This organized, repeating structure comprises paracrystalline tissue spaced 5-15nm apart on the surface of the virus. The production of such fusion proteins will produce multiple ordered, repetitive formations on the surface of the virus. Thus, the resulting ordered, repetitive organization of the first attachment site reflects the normal structure of the native viral protein.

As will be described in more detail in this specification, in another embodiment of the invention, the non-natural molecular scaffold is a recombinant alphavirus, more specifically, a recombinant sindbis virus. Alphaviruses are positive-stranded RNA viruses that replicate their genomic RNA intact in the cytoplasm of infected cells, without the DNA intermediates (Strauss, J. and Strauss, E., Microbiol. Rev.58: 491-562 (1994)). Several members of the alphavirus family, Sindbis virus (Xiong, C. et al, Science 243: 1188-1191 (1989); Schlesinger, S., Trends Biotechnology.11: 18-22(1993)), Semliki Forest Virus (SFV) (Liljester _ m, P. & Garoff, H., Bio/Technology 9: 1356-. Recently, several patents have been issued relating to the use of alphaviruses in heterologous protein expression and vaccine development (see U.S. Pat. Nos. 5,766,602; 5,792,462; 5,739,026; 5,789,245 and 5,814,482). Construction of the alphavirus scaffold of the invention can be carried out using methods well known in the art of recombinant DNA technology, as described in the above articles, which are incorporated herein by reference.

A variety of different recombinant host cells can be utilized to produce virus-based core particles for antigen or antigenic determinant attachment. For example, alphaviruses are known to have a broad host range; sindbis virus infects cultured mammalian, reptile and amphibian cells, as well as certain insect cells (Clark, H., J.Natl.cancer Inst.51: 645 (1973); Leake, C., J.Gen.Virol.35: 335 (1977); Stollar, V., togavirus, R.W.Schleinger eds., Academic Press, (1980), page 583-. Thus, a large number of recombinant host cells may be used in the practice of the present invention. BHK, COS, Vero, HeLa and CHO cells are particularly suitable for the production of heterologous proteins because they have the ability to glycosylate heterologous proteins in a manner similar to human cells (Watson, E. et al, Glycobiology 4: 227, (1994)) and can be selected (Zang, M. et al, Bio/Technology 13: 389(1995)) or genetically engineered (Renner W. et al, Biotech.Bioeng.4: 476 (1995); Lee K. et al, Biotech.Bioeng.50: 336(1996)) for growth in serum-free media and suspensions.

Introduction of a polynucleotide vector into a host cell can be accomplished using methods described in standard Laboratory manuals (see, e.g., molecular cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), Chapter 9; modern methods of molecular biology, John H.Wiley & Sons, Inc. (1997), Chapter 16, written by Ausubel, F. et al), including methods such as electroporation, DEAE-dextran-mediated transfection, microinjection, cationic lipid-mediated transfection, transduction, scraper loading, shock introduction, infection, and the like. U.S. Pat. No. 5,580,859 to Felgner, P. et al describes methods for introducing foreign DNA sequences into host cells.

The host cell may also be infected with the packaged RNA sequence. These packaged RNA sequences can be introduced into host cells by adding them to the culture medium. For example, the preparation of non-infectious alphavirus particles is described in a number of literature sources, including the "sindbis virus expression system", version C (Invitrogen catalog No. K750-1).

When mammalian cells are used as recombinant host cells to produce virus-based core particles, these cells are typically grown in tissue culture. Methods of culturing cells in culture are well known in the art (see, e.g., Celis, cell biology, academic Press, 2 nd edition, (1998); molecular cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor, N.Y. (1989); modern molecular biology, John H.Wiley & Sons, Inc. (1997); Freshney, R., "culture of animal cells," AlR.Liss, Inc. (1983)), Sambrook, J.et al.

It will be appreciated by those skilled in the art that the first attachment site may be, or be part of, any suitable protein, polypeptide, sugar, polynucleotide, peptide (amino acid), natural or synthetic polymer, secondary metabolite or combination thereof that enables the specific binding of the selected antigen or antigenic determinant to the non-natural molecular scaffold. In one embodiment, the attachment site is a protein or peptide that may be selected from a range well known in the art. For example, the first attachment site may be selected from: ligands, receptors, lectins, avidin, streptavidin, biotin, epitopes such as the HA or T7 tag, Myc, Max, immunoglobulin domains, and any other amino acid sequence known in the art to be capable of serving as a first attachment site.

It will also be appreciated by those skilled in the art that in another embodiment of the invention, the first attachment site may be incidentally created in the construction of a former (i.e., a protein or polypeptide) for in-frame fusion with the capsid protein. For example, a protein can be used to fuse with an envelope protein having an amino acid sequence known to be glycosylated in a particular manner, and the added sugar moiety can then serve as the first attachment site for the viral scaffold by binding to a lectin, which serves as the second attachment site for the antigen. Furthermore, the formative sequence may be biotinylated in vivo, the biotin moiety may serve as the first attachment site of the present invention, or the formative sequence may be chemically modified in vitro of various amino acid residues, such modification serving as the first attachment site.

In another embodiment of the invention, the JUN-FOS leucine zipper protein domain fused in-frame to the hepatitis B capsid (core) protein (HBcAg) is selected as the first attachment site. However, it will be clear to all skilled in the art that other viral capsid proteins may be used in the fusion protein construct in order to locate the first attachment site within the non-natural molecular scaffold of the invention.

In another embodiment of the invention, a lysine or cysteine residue fused in-frame to HBcAg is selected as the first attachment site. However, it will be clear to all skilled in the art that other viral capsid proteins or virus-like particles may be used in the fusion protein construct in order to locate the first attachment site within the non-natural molecular scaffold of the present invention.

The JUN amino acid sequence for the first attachment site is as follows:

CGGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVGC(SEQ ID NO：59)

in this case, the expected second attachment site on the antigen is the FOS leucine zipper protein domain, whose amino acid sequence is as follows:

CGGLTDTLQAETDQVEDEKSALQTEIANLLKEKEKLEFILAAHGGC(SEQ ID NO：60)

these sequences are derived from the transcription factors JUN and FOS, each flanked by a short sequence containing cysteine residues at both ends. These sequences are known to be able to interact with each other. The initially assumed structure of the JUN-FOS dimer was that the hydrophobic side chain of one monomer was interlaced with the corresponding side chain of the other monomer in a zipper-like manner (Landshulz et al, Science 240: 1759-. However, this hypothesis has been shown to be erroneous and these proteins are known to form an alpha-helical coiled coil (O 'Shea et al, Science 243: 538-542 (1989); O' Shea et al, Cell 68: 699-708 (1992); Cohen & Parry, Trends biochem. Sci.11: 245-248 (1986)). Thus, the term "leucine zipper" is often used to refer to these protein domains, more for historical reasons than structural reasons. In this patent, the term "leucine zipper" refers to the above-described sequence or a sequence substantially similar to one of the above. The terms JUN and FOS are used to refer to the respective leucine zipper domains, rather than the entire JUN and FOS proteins.

As previously mentioned, the present invention includes a virus-based core particle comprising or consisting of a virus, a virus-like particle, a bacteriophage, a viral capsid particle or a recombinant form thereof. The skilled person knows how to produce such core particles and attach them to the forming body. Examples 17-22 of WO 00/32227, incorporated herein by reference, disclose the production of hepatitis B virus-like particles and measles virus capsid particles as core particles. In these embodiments, the JUN leucine zipper protein domain or FOS leucine zipper protein domain may be formed as a former of the non-natural molecular scaffold of the present invention, thereby serving as a first attachment site.

Examples 23-29 detail the production of hepatitis B core particles carrying an in-frame fusion peptide containing reactive lysine residues and an antigen carrying a genetically fused cysteine residue as the first and second attachment sites, respectively.

In other embodiments, the core particle employed in the compositions of the invention is comprised of hepatitis B capsid (core) protein (HBcAg), fragments of HBcAg, or other proteins or peptides capable of forming an ordered array that have been modified to remove or reduce the number of free cysteine residues. Zhou et al (J.Virol.66: 5393-5398(1992)) demonstrated that modified HBcAg with the removal of naturally occurring cysteine residues retains the ability to bind and form multimeric structures. Thus, core particles suitable for use in the compositions of the invention include core particles comprising a modified HBcAg or fragment thereof wherein one or more of the naturally occurring cysteine residues are deleted or substituted with another amino acid residue (e.g. a serine residue).

HBcAg is a protein produced by processing the hepatitis b core antigen precursor protein. Various isoforms of HBcAg have been identified. For example, a polypeptide comprising SEQ ID NO: 132 is 183 amino acids in length and is produced by processing a 212 amino acid hepatitis B core antigen precursor protein. This processing results in the removal of 29 amino acids from the N-terminus of the hepatitis B core antigen precursor protein. Similarly, a polypeptide comprising SEQ ID NO: 134 is 185 amino acids in length and is produced by processing a precursor protein of hepatitis b core antigen of 214 amino acids. And SEQ ID NO: 132, and the amino acid sequence shown in SEQ ID NO: 134 is represented by SEQ id no: 134 contains a two amino acid insertion at positions 152 and 153.

In most cases, the vaccine compositions of the invention are prepared using a processed form of HBcAg (i.e., HBcAg from which the N-terminal leader sequence of the hepatitis B core antigen precursor protein (e.g., the first 29 amino acid residues shown in SEQ ID NO: 134) has been removed).

Furthermore, when HBcAg is produced under conditions in which processing does not occur, HBcAg is typically expressed in a "processed" form. For example, bacterial systems, such as E.coli, typically do not remove the leader sequence of proteins normally expressed in eukaryotic cells, also referred to as "signal peptides". Thus, when the HBcAg of the present invention is produced using an E.coli expression system, these proteins are usually expressed in a form free from the N-terminal leader sequence of the hepatitis B core antigen precursor protein.

In one embodiment of the invention, the nucleic acid sequence comprising SEQ ID NO: 134 or a portion thereof to prepare a non-native molecular scaffold. In particular, modified hbcags suitable for practicing the invention include the following proteins: wherein the amino acid sequence in a nucleotide sequence corresponding to a nucleotide sequence comprising SEQ ID NO: 134 at positions 48, 61, 107 and 185 of the protein of the amino acid sequence set forth in seq id No. 134 one or more cysteine residues are deleted or substituted with another amino acid residue (e.g., a serine residue). One skilled in the art will recognize that the amino acid sequence of SEQ ID NO: 134, the cysteine residue may also be deleted or substituted with another amino acid residue at a similar position in the different HBcAg variants. Such modified HBcAg variants can then be used to prepare vaccine compositions of the invention.

The invention also includes modified HBcAg variants having deletions or substitutions of one or more of the amino acid sequences set forth in SEQ ID NO: 134, or a cysteine residue not present in the polypeptide of the amino acid sequence shown in seq id No. 134. An example of such an HBcAg variant comprises SEQ ID NO: 90 and 132. These variants have a sequence corresponding to SEQ ID NO: 134 amino acid residue 147 contains a cysteine residue. Accordingly, vaccine compositions of the invention include compositions comprising HBcAg wherein SEQ ID NO: 134 has been deleted.

In some cases (e.g. when heterobifunctional cross-linkers are used to attach antigens or antigenic determinants to non-native molecular scaffolds), it is believed that the presence of free cysteine residues in hbcags results in covalent coupling of toxic components to the core particle, and cross-linking of monomers into undefined forms.

In addition, in many cases, the determination of the compositions of the present invention may not allow the detection of these toxic components. This is because covalent coupling of the toxic component to the non-native molecular scaffold will result in the formation of different products in which the toxic component is attached to, or in some cases does not contain, a different cysteine residue of HBcAg. In other words, each free cysteine residue of each HBcAg is not covalently linked to a toxic component. Furthermore, in many cases, the cysteine residues of a particular HBcAg are not linked to a toxic component. Thus, it can be difficult to detect the presence of these toxic components because they are present in a mixed molecular population. However, administration of HBcAg species containing toxic components to an individual may result in adverse reactions that may be quite severe.

It is well known in the art that free cysteine residues can participate in a variety of chemical side reactions. These side reactions include: disulfide exchange, reaction with, for example, injected or formed chemicals or metabolites in combination therapy with other substances, or direct oxidation, and reaction with nucleotides after exposure to ultraviolet light. This may result in toxic adducts, especially since HBcAg has a strong tendency to bind nucleic acids. With capsids prepared with HBcAg containing free cysteine residues and heterobifunctional crosslinkers, it is difficult to detect such toxic products in antigen-capsid conjugates, as this would result in a product distribution of a wide range of molecular weights. Toxic adducts will therefore be distributed between the various products, which may be present individually in low concentrations, but which when taken together reach toxic levels.

In view of the above, one advantage of using hbcags modified to remove naturally occurring cysteine residues in vaccine compositions is that when an antigen or antigenic determinant is attached to a non-natural molecular scaffold, the sites to which the toxicant is able to bind are reduced in number, or removed altogether. Furthermore, highly modified particles can also be produced with high concentrations of cross-linking agent, which does not have the disadvantage of producing a variety of undefined cross-linked products of HBcAg monomers (i.e. a variety of mixtures of cross-linked monomeric hbcags).

A number of naturally occurring HBcAg variants have been identified as being suitable for use in the present invention. For example, Yuan et al (J.Virol.73: 10122-10128(1999)) describe a polypeptide having a sequence corresponding to SEQ ID NO: 134 with the isoleucine residue at position 97 being replaced with a leucine residue or a phenylalanine residue. The amino acid sequences of various HBcAg variants as well as several hepatitis b core antigen precursor variants are disclosed in the following GenBank report: AAF121240(SEQ ID NO: 89), AF121239(SEQ ID NO: 90), X85297(SEQ ID NO: 91), X02496(SEQ ID NO: 92), X85305(SEQ ID NO: 93), X85303(SEQ ID NO: 94), AF151735(SEQ ID NO: 95), X85259(SEQ ID NO: 96), X85286(SEQ ID NO: 97), X85260(SEQ ID NO: 98), X85317(SEQ ID NO: 99), X85298(SEQ ID NO: 100), AF043593(SEQ ID NO: 101), M20706(SEQ ID NO: 102), X85295(SEQ ID NO: 103), X80925(SEQ ID NO: 104), X85284(SEQ ID NO: 105), X85275(SEQ ID NO: 106), X72702(SEQ ID NO: 107), X85291(SEQ ID NO: 108), X65258(SEQ ID NO: 109), X85302(SEQ ID NO: 321110), SEQ ID NO: 111), SEQ ID NO: 293(SEQ ID NO: 112), X85293(SEQ ID NO: 111), X85315(SEQ ID NO: 113), U95551(SEQ ID NO: 114), X85256(SEQ ID NO: 115), X85316(SEQ ID NO: 116), X85296(SEQ ID NO: 117), AB033559(SEQ ID NO: 118), X59795(SEQ ID NO: 119), X85299(SEQ ID NO: 120), X85307(SEQ ID NO: 121), X65257(SEQ ID NO: 122), X85311(SEQ ID NO: 123), X85301(SEQ ID NO: 124), X85314(SEQ ID NO: 125), X85287(SEQ ID NO: 126), X85272(SFQ ID NO: 127), X85319(SEQ ID NO: 128), AB010289(SEQ ID NO: 129), X85285(SEQ ID NO: 130), AB010289(SEQ ID NO: 131), AF121242(SEQ ID NO: 132), M135 (SEQ ID NO: 135), P031520 (SEQ ID NO: 110999), SEQ ID NO: 110959 (SEQ ID NO: 95138), the disclosures of which are incorporated herein by reference. These HBcAg variants differ in amino acid sequence at multiple positions, including the amino acid sequence corresponding to the sequence located in SEQ ID NO: 134 at amino acid residues 12, 13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149, 157, 176, 178, 182, and 183.

Hbcags suitable for use in the present invention may be derived from any organism so long as they are capable of binding to form an ordered and repetitive antigen array.

As described above, processed hbcags (i.e. hbcags lacking leader sequences) are typically used in the vaccine compositions of the invention. Thus, when using a polypeptide comprising SEQ ID NO: 136. 137 or 138, the N-terminal 30, 35-43 or 35-43 amino acid residues of these proteins are typically removed, respectively.

The invention includes vaccine compositions, and methods of using these compositions, in which the variant hbcags described above are used to prepare non-native molecular scaffolds.

Other HBcAg variants that are capable of binding to form a dimeric or multimeric structure are also within the scope of the invention. Accordingly, the invention also includes a vaccine composition comprising an HBcAg polypeptide comprising or consisting of the amino acid sequence: and SEQ ID NO: 89-132 and 134-138, and processed forms of these proteins where the N-terminal leader sequence is removed, if necessary.

Whether the amino acid sequence of the polypeptide contains a sequence identical to SEQ ID NO: 89-132 and 134-138, or a portion thereof, is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of any one of the sequences shown herein, as determined conventionally using known computer programs such as the Bestfit program. When determining whether a particular sequence is, for example, 95% identical to a reference amino acid sequence of the invention using Bestfit or any other sequence alignment program, the parameters are set such that the percent identity is calculated over the full length of the reference amino acid sequence and gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

Contains SEQ ID NO: the HBcAg variant and the precursor of the amino acid sequences shown in 89-132 and 134-136 are relatively similar to each other. Thus, a sequence corresponding to the sequence of SEQ ID NO: the amino acid residue of the HBcAg variant at position 134 refers to the amino acid sequence set forth in SEQ ID NO: 134 at the position indicated in the amino acid sequence shown in seq id no. The homology between these HBcAg variants is mostly high enough between hepatitis b viruses that can infect mammals, and so one skilled in the art consults SEQ ID NO: 134 and the amino acid sequence of a particular HbcAg variant and there is little difficulty in identifying the "corresponding" amino acid residues. For example, SEQ ID NO: 135, which shows the HBcAg amino acid sequence from a virus infecting woodchuck and a polypeptide comprising SEQ ID NO: 134 has sufficient homology to the HBcAg of the amino acid sequence shown in SEQ ID NO: 134 between amino acid residues 155 and 156 of SEQ ID NO: 135 there is an insertion of 3 amino acid residues.

The amino acid sequence of HBcAg of hepatitis b virus infecting wild geese and ducks is quite different from that of HBcAg of hepatitis b virus infecting mammals, so these protein forms are similar to seq id NO: 134 are difficult to align. However, the invention includes vaccine compositions comprising hepatitis b virus HBcAg variants that infect birds, as well as vaccine compositions comprising fragments of these HBcAg variants. HBcAg fragments suitable for use in preparing the vaccine compositions of the invention include compositions comprising a polypeptide fragment comprising or consisting of amino acid residues selected from the group consisting of: SEQ ID NO: 137, 36-240, 36-269, 44-240, 44-269, 36-305, and 44-305, or SEQ ID NO: 36-240, 36-269, 44-240, 44-269, 36-305, and 44-305 of 138. One skilled in the art will recognize that one, two, three or more cysteine residues naturally occurring in these polypeptides (e.g., the cysteine residues at positions 153 of SEQ ID NO: 137 or 34, 43 and 196 of SEQ ID NO: 138) may be substituted with another amino acid residue, or deleted, prior to inclusion in the vaccine compositions of the present invention.

In one embodiment, the polypeptide comprising SEQ ID NO: 134 is deleted from or substituted with another amino acid residue for the cysteine residues at positions 48 and 107, but the cysteine residue at position 61 is retained. The modified polypeptide is then used to prepare the vaccine composition of the invention.

As described in example 31 below, the solvent accessible cysteine residues at positions 48 and 107 can be removed, for example, by site-directed mutagenesis. The inventors also found that the Cys-48-Ser, Cys-107-Ser HBcAg double mutant constructed as described in example 31 was capable of being expressed in E.coli.

As described above, removal of free cysteine residues reduces the number of sites at which toxic components can bind to HBcAg, and also removes sites at which lysine and cysteine residues of the same or adjacent HBcAg molecules can crosslink. The cysteine at position 61 is involved in dimer formation and forms a disulfide bond with the cysteine at position 61 of another HBcAg, which is normally left intact, to stabilize HBcAg dimers and multimers of the invention.

As described in example 32, crosslinking experiments with (1) HBcAg containing free cysteine residues and (2) HBcAg rendered non-reactive with free cysteine residues using iodoacetamide showed that the free cysteine residues of HBcAg are responsible for crosslinking between HBcAg by reaction between heterobifunctional crosslinker-derived lysine side chains and free cysteine residues. Example 32 also shows that cross-linking of HBcAg subunits results in the formation of high molecular weight products of indeterminate size which cannot be resolved by SDS-polyacrylamide gel electrophoresis.

When the antigen or antigenic determinant is linked to the non-natural molecular scaffold via a lysine residue, a substitution or deletion is made at a position corresponding to the amino acid sequence of SEQ ID NO: either or both of the naturally occurring lysine residues at positions 7 and 96 in 134, as well as other lysine residues present in the HBcAg variant, may be advantageous. Removal of these lysine residues results in the removal of antigen or epitope binding sites that may disrupt the ordered array, and should improve the quality and homogeneity of the final vaccine composition.

In many cases, when the sequence corresponding to SEQ ID NO: 134 at positions 7 and 96, another lysine was introduced into HBcAg as an attachment site for an antigen or antigenic determinant. For example, example 23 below describes a method of inserting such lysine residues. When, for example, the sequence corresponding to SEQ ID NO: 134 at positions 7 and 96, and in an attempt to attach antigens or antigenic determinants to a non-native molecular scaffold using a heterobifunctional crosslinker, it is often advantageous to introduce one lysine residue into the HBcAg.

It has been shown that the C-terminus of HBcAg directs the nuclear localization of the protein (Eckhardt et al, J.Virol.65: 575-582 (1991)). In addition, it is considered that this region of the protein imparts the ability of HBcAg to bind nucleic acid.

In certain embodiments, the vaccine compositions of the invention contain hbcags having nucleic acid binding activity (e.g., contain a naturally occurring HBcAg nucleic acid binding domain). Hbcags comprising one or more nucleic acid binding domains can be used to prepare vaccine compositions with improved T cell stimulatory activity. Accordingly, vaccine compositions of the invention include compositions comprising hbcags having nucleic acid binding activity. Also included are vaccine compositions in which HBcAg is bound to nucleic acid and the use of these compositions in vaccination methods. These hbcags can bind to the nucleic acid prior to administration to the individual, or can bind to the nucleic acid after administration.

In other embodiments, the vaccine compositions of the invention contain HBcAg from which the C-terminal region (e.g., amino acid residues 145-185 or 150-185 of SEQ ID NO: 134) has been removed and which is unable to bind nucleic acid. Thus, additional modified hbcags suitable for use in the present invention include C-terminal truncated mutants. Suitable truncation mutants include hbcags with 1, 5, 10, 15, 20, 25, 30, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 48 amino acids removed from the C-terminus.

Hbcags suitable for use in the present invention also include N-terminal truncation mutants. Suitable truncation mutants include modified hbcags in which 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been removed from the N-terminus.

Hbcags suitable for use in the present invention also include N-terminal and C-terminal truncation mutants. Suitable truncation mutants include hbcags with 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids removed from the N-terminus and 1, 5, 10, 15, 20, 25, 30, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 48 amino acids removed from the C-terminus.

The invention also includes vaccine compositions comprising a HBcAg polypeptide comprising or consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to a truncation mutant as described above.

As described above, in certain embodiments of the invention, a lysine residue is introduced into a polypeptide that forms a non-native molecular scaffold as a first attachment site. In a preferred embodiment, the polypeptide is encoded by a nucleotide sequence comprising or consisting of SEQ ID NO: 134 or amino acids 1-144 or amino acids 1-149 the sequence modified such that the amino acids corresponding to positions 79 and 80 are substituted with a peptide having a Gly-Lys-Gly-amino acid sequence (seq id NO: 158), the cysteine residues at positions 48 and 107 are deleted or substituted with another amino acid residue, while the cysteine at position 61 is retained. The invention also includes vaccine compositions comprising a corresponding fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 89-132 and 135-136, and also contain the amino acid changes described above.

The invention also includes vaccine compositions comprising a HBcAg fragment comprising or consisting of a sequence other than SEQ ID NO: 134, wherein in SEQ ID NO: 134 have been deleted for cysteine residues not present at the corresponding positions. An example of such a fragment is a fragment comprising or consisting of SEQ ID NO: 132, wherein the cysteine residue at position 147 is replaced with another amino acid residue or is deleted.

The invention also includes vaccine compositions comprising an HBcAg polypeptide comprising or consisting of the amino acid sequence: and SEQ ID NO: 134, and amino acids 1-144 or 1-149 comprising SEQ ID NO: 89-132 or 134-136, and the corresponding portion of any one of the amino acid sequences set forth in SEQ ID NO: 158 or 1-152 is at least 80%, 85%, 90%, 95%, 97% or 99% identical.

The invention also includes vaccine compositions comprising an HBcAg polypeptide comprising or consisting of the amino acid sequence: and SEQ ID NO: 137, amino acids 36-240, 36-269, 44-240, 44-269, 36-305, and 44-305 of SEQ ID NO: 138, amino acids 36-240, 36-269, 44-240, 44-269, 36-305 and 44-305 are at least 80%, 85%, 90%, 95%, 97% or 99% identical.

The vaccine composition of the invention may comprise a mixture of different hbcags. Thus, these vaccine compositions may consist of hbcags differing in amino acid sequence. For example, vaccine compositions may be prepared containing "wild-type" HBcAg and modified HBcAg in which one or more amino acid residues are altered (e.g.deleted, inserted or substituted). However, in most applications only one type of HBcAg, or at least a plurality of hbcags containing substantially the same first attachment site, is used because vaccines prepared with these hbcags present a highly ordered, repetitive antigen or antigenic determinant array. Furthermore, preferred vaccine compositions of the invention are compositions that exhibit a highly ordered, repetitive antigen array.

The invention also includes vaccine compositions in which the non-native molecular scaffold is prepared using HBcAg fused to another protein. As mentioned above, other examples of HBcAg fusion proteins suitable for use in the vaccine composition of the invention include fusion proteins to which amino acid sequences are added that aid in the formation and/or stabilization of HBcAg dimers and multimers. The additional amino acid sequence may be fused to the N-terminus or C-terminus of HBcAg. An example of such a fusion protein is the fusion of HBcAg with the helical region of Saccharomyces cerevisiae GCN4 (GenBank accession number P03069(SEQ ID NO: 154)).

The helical domain of the GCN4 protein forms homodimers through non-covalent interactions, which can be used to make and stabilize HBcAg dimers and multimers.

In one embodiment, the invention provides a vaccine composition prepared using an HBcAg fusion protein comprising an HBcAg or fragment thereof, and a peptide comprising SEQ id no: a GCN4 polypeptide having the sequence of amino acid residue 227 and 276. The GCN4 polypeptide may also be fused to the N-terminus of HBcAg.

The HBcAg/src homology 3(SH3) domain fusion protein may also be used to prepare vaccine compositions of the invention. The SH3 domain is a relatively small domain found in a variety of proteins that provides the ability to interact with specific proline-rich sequences in protein binding partners (see McPherson, Cell Signal 11: 229-238 (1999)). The HBcAg/SH3 fusion protein can be used in several ways. First, SH3 domains are capable of forming a first attachment site for interacting with a second attachment site for an antigen or antigenic determinant. Similarly, another proline-rich amino acid sequence may also be added to HBcAg and used as a first attachment site for the SH3 domain second attachment site of an antigen or antigenic determinant. Second, the SH3 domain binds to a proline-rich region introduced into HBcAg. Thus, SH3 domains and proline-rich SH3 interaction sites can be inserted into the same or different hbcags to form and stabilize the dimers and multimers of the invention.

In other embodiments, the vaccine composition of the invention is prepared using a bacterial pilin protein, a bacterial pilin portion, or a fusion protein containing a bacterial pilin protein or portion thereof. Examples of pilin proteins include those produced by Escherichia coli, Haemophilus influenzae (Haemophilus influenzae), Neisseria meningitidis (Neisseria meningitidis), Bacillus crescentus (Caulobacter rcrentus), Pseudomonas stutzeri (Pseudomonas stutzeri), and Pseudomonas aeruginosa (Pseudomonas aeruginosa). Amino acid sequences of pilin proteins suitable for use in the present invention include those described in GenBank reports AJ000636(SEQ ID NO: 139), AJ132364(SEQ ID NO: 140), AF229646(SEQ ID NO: 141), AF051814(SEQ ID NO: 142), AFO51815(SEQ ID NO: 143) and X00981(SEQ ID NO: 155), the complete disclosures of which are incorporated herein by reference.

Bacterial pilin proteins are typically processed to remove the N-terminal leader sequence before the protein is exported into the bacterial periplasm. One skilled in the art will recognize that the bacterial pilin proteins used to prepare the vaccine compositions of the present invention are generally free of naturally occurring leader sequences.

A specific example of a pilin suitable for use in the present invention is the P-pilin of E.coli (GenBank report AF237482(SEQ ID NO: 144)). An example of a type 1 E.coli pilin suitable for use in the present invention is one comprising the amino acid sequence (SEQ ID NO: 146) described in GenBank report P04128, encoded by a nucleic acid having the nucleotide sequence (SEQ ID NO: 145) described in GenBank report M27603. The complete disclosures of these GenBank reports are incorporated herein by reference. The vaccine compositions of the present invention are generally prepared using the mature forms of the above proteins.

Bacterial pilin or pilin moieties suitable for use in the present invention are generally capable of binding to form a non-natural molecular scaffold.

Methods for the preparation of pili and pilus-like structures in vitro are known in the art. For example, Bullitt et al, proc.natl.acad.sci.usa93: 12890-12895(1996) describes the in vitro reconstitution of the P-pilus subunit of E.coli. In addition, Eshdat et al, j.bacteriol.148: 308-314(1981) describe methods suitable for dissociating E.coli type 1 pili and pili reconstruction. Briefly, these methods are as follows: the pili were dissociated by incubation in saturated guanidine hydrochloride at 37 ℃. The pilin was then purified by chromatography, followed by dialysis against 5mM tris (hydroxymethyl) aminomethane hydrochloride (pH8.0) to form a pilin dimer. Eshdat et al also found that the compounds contained 5mM MgCl₂Dialyzed against 5mM Tris (hydroxymethyl) aminomethane (pH8.0), the pilin dimers reassembled to form pili.

In addition, the pilin protein can be modified to contain a first attachment site to which an antigen or antigenic determinant is linked via a second attachment site, for example, using conventional genetic engineering and protein modification methods. Alternatively, the antigen or antigenic determinant can also be linked directly to the naturally occurring amino acid residues in these proteins via a second attachment site. These modified pilin proteins can then be used in the vaccine composition of the invention.

The bacterial pilins used to prepare the vaccine compositions of the invention may be modified in a manner similar to that described herein for HBcAg. For example, cysteine and lysine residues may be deleted, or may be substituted with other amino acid residues, and a first attachment site may be added to these proteins. Furthermore, the pilin may be expressed in modified form, or may be chemically modified after expression. Similarly, whole pili can be harvested from bacteria and then chemically modified.

In another embodiment, pili or pilus-like structures are harvested from bacteria (e.g., E.coli) for use in the composition of the vaccine of the invention. An example of a pilus suitable for preparing a vaccine composition is an e.coli type 1 pilus, which is a pilus consisting of a pilus having the sequence of SEQ ID NO: 146, or a pilin monomer of the amino acid sequence shown in seq id no.

Various methods of harvesting bacterial pili are known in the art. For example, Bullitt and Makowski (Biophys. J.74: 623-632(1998)) describe a pilus purification method for harvesting P-pili from E.coli. According to this method, the pili are sheared from E.pilus containing P-pilus plasmids by lysis and MgCl₂(1.0M) Recycling purification of the precipitate. A similar purification procedure is described in example 33 below.

After harvesting, the pili or pilus-like structures can be modified in a variety of ways. For example, a first attachment site may be added to the pilus, and an antigen or antigenic determinant may bind thereto via a second attachment site. In other words, the bacterial pili or pilus-like structures can be harvested and modified to form a non-natural molecular scaffold.

The pilus or pilus-like structure may also be modified by attachment of an antigen or antigenic determinant in the absence of non-natural formers. For example, an antigen or antigenic determinant may be linked to a naturally occurring cysteine residue or lysine residue. In this case, the high order and reproducibility of the naturally occurring amino acid residues will direct the coupling of antigens or antigenic determinants to pili or pilus-like structures. For example, a pilus or pilus-like structure can be attached to the second attachment site of an antigen or antigenic determinant using a heterobifunctional cross-linking agent.

When preparing the vaccine compositions of the present invention using naturally synthesized structures of organisms (e.g., pili), it is often advantageous to genetically construct these organisms such that they produce structures having the desired characteristics. For example, when using E.coli type 1 pili, the E.coli from which the pili are to be harvested can be modified in order to produce a structure with the desired characteristics. Examples of possible modifications of pilin include insertion of one or more lysine residues, deletion or substitution of one or more naturally occurring lysine residues, and deletion or substitution of one or more naturally occurring cysteine residues (e.g., cysteine residues at positions 44 and 84 in SEQ ID NO: 146).

Other modifications of the pilin gene may also be made to produce expression products containing a first attachment site (e.g., FOS or JUN domain) other than a lysine residue. Of course, suitable first attachment sites are generally limited to those that do not prevent the pilin protein from forming a pilus or pilus-like structure suitable for the vaccine composition of the invention.

The pilin genes naturally present in bacterial cells may be modified in vivo (e.g., by homologous recombination), or pilin genes having particular characteristics may be inserted into these cells. For example, the pilin gene can be introduced into a bacterial cell as part of a replicable cloning vector or a vector that can be inserted into a bacterial chromosome. The inserted pilin gene may also be linked to an expression regulatory control sequence (e.g., the lac operon).

In most cases, the pili or pilus-like structure used in the vaccine composition of the invention consists of one pilin subunit. Pili or pilus-like structures composed of identical subunits are generally used because they are expected to form structures that exhibit highly ordered, repetitive antigen arrays.

However, the compositions of the present invention also include vaccines comprising pili or pilus-like structures composed of different species of pilin subunits. The pilin subunits that make up these pili or pilus-like structures can be expressed from genes that occur naturally in the bacterial cell, or the genes can be introduced into the cell. When expressing naturally occurring pilin genes and introduced genes in cells that form pili or pilus-like structures, the product is typically a structure consisting of a mixture of these pilins. In addition, when a bacterial cell expresses two or more pilin genes, the relative expression of the various pilin genes is generally a factor in determining the ratio of different pilin subunits in a pilus or pilus-like structure.

When it is desired to obtain a pilus or pilus-like structure having a particular mixed pilin subunit composition, a heterologous inducible promoter may be used to regulate the expression of at least one pilin gene. Such promoters, as well as other genetic elements, can be used to regulate the relative amounts of different pilin subunits in bacterial cells, and thus the composition of pili or pilus-like structures.

In addition, in most cases, where an antigen or antigenic determinant is linked to a bacterial pilus or pilus-like structure by a bond other than a peptide bond, bacterial cells producing the pilus or pilus-like structure used in the compositions of the invention can be genetically engineered to produce a pilus protein fused to the antigen or antigenic determinant. These fusion proteins that form pili or pilus-like structures are suitable for use in the vaccine compositions of the invention.

As described above, the viral capsid can be used for (1) presentation of an antigen or antigenic determinant, and (2) preparation of the vaccine composition of the invention. Among the viral capsid proteins useful in the practice of the present invention are those, also referred to herein as "coat proteins," which form a capsid or capsid-like structure upon expression. Thus, these capsid proteins can form the core particle and the non-native molecular scaffold. These capsids or capsid-like structures generally form an ordered and repetitive array that can be used for the presentation of antigens or antigenic determinants and the preparation of the vaccine compositions of the invention.

Various methods can be used to attach one or more (e.g., 1, 2, 3, 4, 5, etc.) antigens or antigenic determinants to one or more (e.g., 1, 2, 3, 4, 5, etc.) proteins that form capsids or capsid-like structures (e.g., bacteriophage coat proteins) and other proteins. For example, the first and second attachment sites may be used to attach an antigen or antigenic determinant to the core particle. In addition, one or more (e.g., 1, 2, 3, 4, 5, etc.) heterobifunctional crosslinkers can be used to attach antigens or antigenic determinants to one or more proteins that form a viral capsid or capsid-like structure.

For example, the core particles and vaccine compositions of the invention may be prepared using viral capsid proteins or fragments thereof. The bacteriophage Q β coat protein may be expressed recombinantly in e.coli, for example. Moreover, these proteins form capsids automatically upon expression. Moreover, these capsids form an ordered and repetitive antigen or antigenic determinant array, which can be used for antigen presentation and vaccine composition preparation. As described in example 38 below, bacteriophage Q β coat protein may be used to prepare vaccine compositions that elicit an immune response to an antigenic determinant.

Specific examples of phage coat proteins that can be used to prepare the compositions of the invention include coat proteins of the following phages: RNA bacteriophages, such as bacteriophage Q beta (SEQ ID NO: 159, accession number VCBPQ beta for PIR database of Q beta CP, and SEQ ID NO: 217, accession number AAA16663 for Q beta A1 protein), bacteriophage R17(SEQ ID NO: 160; PIR accession number VCBPR7), bacteriophage fr (SEQ ID NO: 161; PIR accession number VCBPFR), bacteriophage GA (SEQ ID NO: 162; GenBank accession number NP-040754), bacteriophage SP (SEQ ID NO: 163, Bank accession number CAA30374 for SP CP, and SEQ ID NO: 254, accession number for SPA1 protein), bacteriophage MS2(SEQ ID NO: 164; PIR accession number VCBPM2), bacteriophage M11(SEQ ID NO: 165; GenBank accession number AAC06250), bacteriophage MX1(SEQ ID NO: 146166; GenBank accession number AAC 99), bacteriophage NL95(SEQ ID NO: 167; GenBank accession number AAC 14732), bacteriophage AAC 0332; SEQ ID NO: 03 2; SEQ ID NO: 0336215; SEQ ID NO: 03 2; SEQ ID NO: 033675; SEQ ID NO: 033699; SEQ ID NO: 254, SEQ ID NO: 0324), Phage PP7(SEQ ID NO: 253). One skilled in the art will recognize that any protein that can form a capsid or capsid-like structure can be used in the preparation of the vaccine compositions of the invention. Furthermore, the a1 protein or a C-terminal truncated form lacking up to 100, 150 or 180 amino acids at the C-terminus of bacteriophage Q β may be incorporated in capsid assembly of Q β coat protein. The a1 protein may also be fused to a former, and thus to the first attachment site, for attachment of an antigen containing the second attachment site. To ensure capsid formation, the percentage of a1 protein relative to Q β CP in the capsid assembly is limited. The A1 protein has accession number AAA16663(SEQ ID NO: 217).

It has also been found that Q β coat protein self-assembles into a capsid when expressed in E.coli (Kozlovska TM. et al, GENE 137: 133-137 (1993)). The obtained capsids or virus-like particles show an icosahedral phage-like capsid structure with a diameter of 25nm and a T-3 quasisymmetry. And the crystal structure of bacteriophage Q β has been resolved. The capsid contains 180 copies of the coat protein, which are linked by disulfide bonds in the form of covalent pentamers and hexamers (Golomahmmadi, R. et al, Structure 4: 543-. Other RNA phage coat proteins have also been shown to self-assemble following expression in bacterial hosts (Kastelein, RA. et al, Gene 23: 245-. The Q β phage capsid contains, in addition to the coat protein, the so-called readthrough protein a1 and the mature protein a 2. A1 was generated by suppression at the UGA stop codon and was 329 amino acids in length. The capsid of the recombinant coat protein of bacteriophage Q β used in the present invention lacks the a2 cleavage protein and contains RNA from the host. The coat protein of RNA phages is an RNA binding protein that interacts with the stem-loop of the ribosome binding site of the replicase gene and acts as a translational repressor of the viral life cycle. The sequence and structural components of this interaction are known (Witherll, GW. & Uhlenbeck, OC. biochemistry 28: 71-76 (1989); Lim F. et al, J. biol. chem. 271: 31839-31845 (1996)). It is known that stem loops and RNA are commonly involved in viral assembly (Golomammadi, R. et al, Structure 4: 543-.

The effect of the protein or protein domain on particle structure and assembly is greater than that of the short peptide. As an example, when Q β particles are expressed, it is generally not possible for the disulfide-containing antigen to fold correctly in the cytoplasm of e. Also, glycosylation is generally not possible in prokaryotic expression systems. Thus, attaching antigen to particles starting from already assembled particles and isolated antigen is an advantage of the invention described herein. This allows the particles and antigen to be expressed in an expression host, ensuring proper folding of the antigen and proper folding and assembly of the particles.

One discovery of the present invention is that one or several antigenic molecules can be attached to a subunit of the capsid of the RNA bacteriophage coat protein. A particular feature of the capsid of RNA bacteriophage coat proteins, in particular of the Q β capsid, is the possibility of coupling several antigens per subunit. This can result in a dense array of antigens. Other viral capsids covalently bound to antigen by chemical cross-linking, such as HBcAg modified by one lysine residue in the major immunodominant region (MIR; WO 00/32227), show coupling densities of up to 0.5 antigens per subunit. The distance between spikes of HBcAg (corresponding to MIR) was 50A (Wynne, SA. et al, mol. cell 3: 771-780(1999)), and therefore antigen arrays with a spacing of less than 50A could not be generated.

The capsid of Q β coat protein displays a defined number of lysine residues on its surface, with a defined topology, 3 lysine residues pointing to the inside of the capsid and interacting with RNA, and the other 4 lysine residues exposed to the outside of the capsid. These defined characteristics facilitate the attachment of the antigen to the outside of the particle, rather than the inside where the lysine residues interact with the RNA. The capsids of other RNA phage coat proteins also have a defined number of lysine residues on their surface, which also have a defined topology. Capsids derived from RNA bacteriophages have the further advantage of high expression levels in bacteria, which allows the production of large amounts of material at an economical cost.

Another characteristic of the capsid of Q β coat protein is its stability. The Q beta subunits are covalently linked to each other by disulfide bonds. Q β capsid protein also shows extraordinary resistance to organic solvents and denaturants. We have surprisingly seen that DMSO and acetonitrile concentrations of up to 30% and guanidine salt concentrations of up to 1M can be employed without affecting capsid stability or the ability to form antigen arrays. Thus, hydrophobic peptides can be coupled with these organic solvents. The high stability of the Q β coat protein capsid is an important feature, particularly useful for immunization and vaccination of mammals and humans. The resistance of the capsid to organic solvents enables coupling to antigens that are insoluble in aqueous buffers.

The insertion of cysteine residues into the N-terminal beta hairpin of the coat protein of RNA phage MS-2 has been described in patent application US/5,698,424. However, we note that the presence of exposed free cysteine residues in the capsid can lead to oligomerization of the capsid by forming disulfide bonds. Other attachments described in patent application US/5,698,424 include the formation of disulfide bonds between the antigen and the Q β particles. Thiol-containing molecules are susceptible to such attachment.

The reaction between the existing disulfide bond formed by the cysteine residues on Q β and the antigen containing free thiol residues releases the non-antigenic thiol-containing material. These newly formed thiol-containing species are able to react again with other disulfide bonds present on the particle, thereby establishing an equilibrium. In reacting with the disulfide bonds formed on the particles, the antigen may form disulfide bonds with cysteine residues from the particles or with cysteine residues of the leaving group molecules forming the original disulfide bonds on the particles. Furthermore, the other attachment methods described, utilize a heterobifunctional cross-linker that reacts with cysteine on the Q β particle at one end and lysine residues on the antigen at the other end, resulting in random orientation of the antigen on the particle.

We also note that, unlike the capsid of Q β and Fr coat proteins, recombinant MS-2 described in patent application US/5,698,424 is essentially free of nucleic acid, whereas RNA is encapsulated within both capsids.

We describe novel compositions of the invention that can form strong antigen arrays with variable antigen density. We demonstrate that much higher epitope densities can be achieved than are typically obtained with other VLPs. We also disclose compositions that display several antigens simultaneously at appropriate intervals, and compositions that add accessory molecules, enhance solubility, or modify the capsid in a suitable and desirable manner.

The preparation of capsid compositions of RNA bacteriophage coat proteins with high epitope density is disclosed in the present application. Those skilled in the art will appreciate that the conditions under which an ordered, repetitive antigen array is assembled will depend, in part, on the choice of antigen and second attachment site on the antigen. In the absence of a useful second attachment site, such a second attachment site must be constructed for the antigen.

One prerequisite for the design of a second attachment site is the choice of fusion, insertion or, in general, engineered position. The skilled person knows how to obtain guidance for selecting the location of the second attachment site. The crystal structure of the antigen may provide information about the accessibility of the C-or N-terminus of the molecule (e.g. depending on their accessibility to solvents) or about the exposure of residues suitable as second attachment sites (e.g. cysteine residues) to solvents. The exposed disulfide bonds, as in the case of Fab fragments, can also be the source of the second attachment site, since they are usually converted to a cysteine residue by mild reduction. Where autoantigen immunization is intended to inhibit the interaction of such autoantigen with its natural ligand, a second attachment site is typically added in order to generate antibodies against the interaction site with the natural ligand. Thus, the position of the second attachment site is chosen to avoid steric hindrance of the second attachment site or any amino acid linker containing the second attachment site. In other embodiments, it is desirable for an antibody response to occur against a site other than the interaction site of the autoantigen with its natural ligand. In these embodiments, the second attachment site may be selected so as to prevent the production of antibodies directed against the interaction site of the autoantigen with its natural ligand.

Other criteria for selecting the location of the second attachment site include the oligomerization state of the antigen, the oligomerization site, the presence of cofactors and the presence or absence of experimental evidence revealing sites in the structure and sequence of the antigen, where modification of the antigen is consistent with the function of the autoantigen or with the production of antibodies that recognize the autoantigen.

In certain embodiments, constructing a second attachment site on an antigen entails fusing an amino acid linker that contains an amino acid suitable as the second attachment site in accordance with the present disclosure. In a preferred embodiment, the amino acid is cysteine. Amino acid linkerThe choice of (a) depends on the nature of the autoantigen, its biochemical properties, such as pI, charge distribution, glycosylation. Generally flexible amino acid linkers are advantageous. Examples of amino acid linkers are the hinge region of an immunoglobulin, the glycine serine linker (GGGGS)_nAnd glycine joint (G)_nAll of them contain a cysteine residue as second attachment site and optionally also a glycine residue. The following are examples of such amino acid linkers:

n-terminal γ 1: CGDKTHTSPP

C-terminal gamma 1: DKTHTSPPCG

N-terminal γ 3: CGGPKPSTPPGSSGGAP

C-terminal gamma 3: PKPSTPPGSSGGAPGGCG

An N-terminal glycine linker: GCGGGGG

C-terminal glycine linker: GGGGCG

For peptides, a GGCG linker at the C-terminus or a CGG at the N-terminus of the peptide has been shown to be useful. A glycine residue is typically inserted between the bulky amino acid and the cysteine that serves as the second attachment site.

A particularly preferred method of attaching an antigen to a VLP, particularly to the capsid of a coat protein of an RNA bacteriophage, is to link a lysine residue on the surface of the capsid of the coat protein of the RNA bacteriophage to a cysteine residue on the antigen. To be effective as a second attachment site, the sulfhydryl group must be available for conjugation. Thus, the cysteine residue must be in a reduced state, i.e. there must be a cysteine or cysteine residue containing a free thiol group available. In the case where the cysteine residue as the second attachment site is in oxidized form, if it is to form a disulfide bond, for example, it is necessary to reduce the disulfide bond with, for example, DTT, TCEP or β -mercaptoethanol.

One discovery of the present application is that by selecting the cross-linking agent and other reaction conditions, the epitope density on the coat protein of RNA phage coat can be modulated. For example, the crosslinkers Sulfo-GMBS and SMPH can achieve higher epitope density than the crosslinker Sulfo-MBS under the same reaction conditions. High concentrations of reactants have a positive effect on derivatization and the amount of antigen coupled to the RNA phage coat protein, in particular to the Q β capsid protein, can be controlled by manipulating the reaction conditions.

According to theoretical calculations, the maximum number of globular protein antigens of 17kDa in size can be reached not exceeding 0.5. Thus, several lysine residues of the Q β coat protein capsid will be derivatized by the crosslinker molecule, but will not contain the linked antigen. This results in the disappearance of the positive charge, which may be detrimental to the solubility and stability of the conjugate. By replacing certain lysine residues with arginine as in the disclosed Q β coat protein mutants, we prevented excessive disappearance of positive charges because the arginine residues did not react with the cross-linking agent.

In other embodiments, we disclose a Q β mutant coat protein containing additional lysine residues, which is suitable for obtaining high density antigen arrays.

The crystal structures of several RNA phages have been determined (Golomahmmadi, R. et al, Structure 4: 543-. Using this information, one skilled in the art can readily identify surface exposed residues and modify phage coat proteins to insert one or more reactive amino acid residues. Thus, one skilled in the art can readily produce and identify modified forms of phage coat proteins that can be used in the practice of the present invention. Thus, the vaccine compositions of the invention can also be prepared using protein variants that form capsids or capsid-like structures (e.g., coat proteins of bacteriophage Q β, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophage MS 2).

Although the sequences of the above variant proteins differ from their wild-type counterparts, these variant proteins generally retain the ability to form capsids or capsid-like structures. Thus, the invention also includes vaccine compositions comprising protein variants that form capsids or capsid-like structures, as well as methods of making such vaccine compositions, individual protein subunits used to make such vaccine compositions, and nucleic acid molecules encoding such protein subunits. Thus, variant forms of wild-type proteins that form ordered, repetitive antigen arrays (e.g., protein variants that form capsids or capsid-like structures) and retain the ability to bind to and form capsids or capsid-like structures are included within the scope of the invention.

Thus, the invention also includes vaccine compositions comprising a protein comprising or alternatively consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to a wild-type protein that can form an ordered array. In many cases, it is desirable to process these proteins to remove a signal peptide (e.g., a heterologous signal peptide).

Also included within the scope of the invention are nucleic acid molecules encoding the proteins used to prepare the vaccine compositions of the invention.

In particular embodiments, the invention also includes vaccine compositions comprising a protein comprising or consisting of a sequence identical to SEQ ID NO: 159 and 167 or a processed form of these proteins in which the N-terminal leader sequence has been removed if necessary.

Proteins suitable for use in the present invention also include C-terminal truncation mutants of proteins that can form capsids or capsid-like structures, as well as other ordered arrays. Specific examples of such truncation mutants include those having the amino acid sequence of SEQ ID NO: 159 and 167, wherein 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been removed from the C-terminus. The C-terminal truncation mutants used in the practice of the present invention generally retain the ability to form a capsid or capsid-like structure.

Proteins suitable for use in the present invention also include N-terminal truncation mutants of proteins that can form capsids or capsid-like structures. Specific examples of such truncation mutants include those having the amino acid sequence of SEQ ID NO: 159 and 167, wherein 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been removed from the N-terminus. N-terminal truncation mutants used in the practice of the present invention generally retain the ability to form a capsid or capsid-like structure.

Other proteins suitable for use in the present invention also include N-terminal and C-terminal truncation mutants of proteins that can form capsids or capsid-like structures. Suitable truncation mutants include those having SEQ ID NO: 159 and 167, wherein 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been removed from the N-terminus and 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been removed from the C-terminus. The N-terminal and C-terminal truncation mutants used in the practice of the present invention generally retain the ability to form capsids or capsid-like structures.

The invention also includes vaccine compositions comprising a protein comprising or alternatively consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to the truncation mutants described above.

The invention thus includes vaccine compositions prepared from proteins that can form ordered arrays, methods of preparing vaccine compositions from individual protein subunits, methods of preparing such individual protein subunits, nucleic acid molecules encoding such subunits, and methods of vaccination and/or eliciting an immune response in an individual using the vaccine compositions of the invention.

B. Construction of an antigen or antigenic determinant with a second attachment site

The second component of the composition of the invention is an antigen or antigenic determinant having at least one second attachment site capable of binding to the first attachment site of the non-natural molecular scaffold via at least one non-peptide bond. The present invention provides different compositions depending on the antigen or antigenic determinant selected for the desired therapeutic effect. Other compositions may be provided by altering the molecule selected for the second attachment site.

However, when the vaccine composition of the invention is prepared with bacterial pili or pilus-like structures, pilin, the antigen or antigenic determinant may bind to the pilin by expression of a pilin/antigen fusion protein. Similarly, when a vaccine composition of the invention is prepared with proteins other than pilins (e.g., viral capsid proteins), antigens or antigenic determinants can bind to these non-pilins through expression of the non-pilin/antigen fusion protein. Antigens or antigenic determinants can also be bound by non-peptide bonds to bacterial pili, pilus-like structures, pilin proteins, and other proteins that can form ordered arrays.

The antigen of the invention may be selected from: (a) a protein suitable for eliciting an immune response against cancer cells; (b) proteins suitable for eliciting an immune response against infectious diseases; (c) a protein suitable for eliciting an immune response against an allergen; (d) a protein suitable for eliciting an immune response in livestock; (e) (ii) a fragment (e.g., domain) of any one of the proteins (a) - (d).

In a particular embodiment of the invention, the antigen or antigenic determinant may be used for the prevention of infectious diseases. Such treatment methods are useful for treating a variety of infectious diseases affecting a variety of hosts (e.g., human, bovine, ovine, porcine, canine, feline, other mammalian and non-mammalian species). Infectious diseases that can be treated are well known to those skilled in the art, and examples include: infections with viral pathogens such as HIV, influenza, herpes, viral hepatitis, EB, polio, viral encephalitis, measles, chickenpox, etc.; or infection by bacterial pathogens such as pneumonia, tuberculosis, syphilis, etc.; or infection by parasitic pathogens such as malaria, trypanosomiasis, leishmaniasis, trichomoniasis, amebiasis, etc. Thus, those antigens or antigenic determinants selected for the compositions of the invention are well known in the medical arts; examples of antigens or antigenic determinants include: HIV antigens gp140 and gp 160; influenza antigens hemagglutinin, M2 protein and neuraminidase, hepatitis b surface antigen, circumsporozoite protein of malaria.

In particular embodiments, the present invention provides vaccine compositions suitable for use in methods of preventing and/or attenuating diseases or conditions caused or exacerbated by "self" gene products (e.g., tumor necrosis factor). Thus, vaccine compositions of the invention include compositions that result in the production of antibodies that can prevent and/or attenuate diseases or conditions caused or exacerbated by "self" gene products. Examples of such diseases or conditions include: graft versus host disease, IgE-mediated allergy, adult respiratory distress syndrome, Crohn's disease, allergic asthma, Acute Lymphocytic Leukemia (ALL), non-Hodgkin's lymphoma (NHL), Graves 'disease, Systemic Lupus Erythematosus (SLE), inflammatory autoimmune disease, myasthenia gravis, lymphonodopathy of immunoproliferative disease (IPL), lymphadenopathy of vascular immunoproliferation (AIL), immunoblastic lymphadenopathy (IBL), rheumatoid arthritis, diabetes, multiple sclerosis, Alzheimer's disease and osteoporosis.

In related embodiments, the compositions of the invention are immunotherapeutic agents useful for treating allergy or cancer.

Those skilled in the medical arts of treating allergies know how to select antigens or antigenic determinants for use in preparing compositions and methods of treating allergies. Representative examples of such antigens or antigenic determinants include: bee venom phospholipase A₂Bet vI (birch pollen allergen), 5Dol mV (whitefly wasp venom allergen), melittin and Der pI (dermatophagoides pteronyssinus allergen) and fragments thereof capable of eliciting an immune response.

As mentioned above, a preferred antigen or antigenic determinant is Der pI. Der pI is a 25kD protease found in house dust mite fecal particles. Der pI is the major allergic molecule of house dust mite. Thus, 80% of mite allergy patients contain anti-Der pI IgE antibodies. In particular, the peptides p52-72 and p117-133, etc., are known to contain epitopes recognized by natural Der pI-specific antibodies. IgE antibodies produced in a polyclonal response to intact antigens bind with high affinity to the peptide regions 59-94 (L. Pierson-Mulleny et al, (2000) molecular immunology). Other regions may also bind IgE with high affinity. The peptide p117-133 contains a free cysteine at the N-terminus, preferably as a second attachment site for the present invention. The 3D model distributes the peptides p52-72 and p117-133 on the surface of the whole protein. However, other fragments of the Der pI protein may contain B cell epitopes preferred for use in the present invention.

Those skilled in the medical arts of treating cancer know how to select antigens or antigenic determinants for use in preparing compositions and methods of treating cancer. Representative examples of such antigens or antigenic determinants include: her2 (breast cancer), GD2 (neuroblastoma), EGF-R (malignant glioblastoma), CEA (medullary thyroid carcinoma) and CD52 (leukemia), human melanoma protein gp100, human melanoma protein melan-A/MART-1, tyrosinase, NA17-Ant protein, MAGE-3 protein, p53 protein, HPV 16E 7 protein, and fragments thereof capable of eliciting an immune response. Additional preferred antigenic determinants that can be used in the compositions and methods for treating cancer are molecules and antigenic determinants involved in angiogenesis. Angiogenesis-the formation of new blood vessels-plays an important role in physiological and pathophysiological processes such as wound healing and solid tumor growth, respectively (Folkman, J. (1995) Nat. Medicine1, 27-31; Folkman, J. and Klagsbrun, M. (1987) Science235, 442-. Rapidly growing tumors begin and rely on the formation of blood vessels to provide the required blood supply. Thus, anti-angiogenic agents may be effective anti-cancer treatments.

Among the several putative angiogenic factors identified to date, Vascular Endothelial Growth Factor (VEGF), a potent endothelial cell-specific mitogen, is a major stimulator of angiogenesis in a variety of solid tumors. Although recent findings suggest that a panel of angiogenic factors must fit perfectly to form functional blood vessels, blockade of even one growth factor appears to limit disease-induced vascular growth. Thus, VEGF blockade may be the primary intervention target in tumor-induced angiogenesis. Recently targeting the endothelium rather than the tumor itself has been used as a new strategy for treating tumors (Millauer, b., Shawver, l.k., Plate, k.h., Risau, w. and Ullrich, a. (1994) Nature 367, 576-579; Kim, j., Li, b., Winer, j., armaini, m., Gillett, N., Phillip, h.s., Ferrara, N. (1993) Nature 362, 841-844). Unlike tumors, where the target structures recognized by the immune system are susceptible to mutation, endothelial cells are often unable to escape the immune system or other treatment protocols.

An anti-VEGFR-II antibody (IMC-1C11) and an anti-VEGF antibody (Lu, D., Kusie, P., Pytowski, B., Persaud, K., Bohlen, P., Witte, L., Zhu, Z. (2000) J.biol.chem.275, 14321. congol 14330; Presta, L.G., Chen, H., O' Connor, SJ., Chisholm, V., Meng, YG., Krummen, L., Winkler, M., Ferrara N. (1997) Cancer Res.47, 4593. congol 4599) have been disclosed. The former neutralizing monoclonal anti-VEGFR-2 antibody recognizes an epitope identified as a putative VEGF/VEGFR-II binding site (Piosek, C., Schneider-Mergener, J., Schirener, M., Vakalopoulou, E., Germeroth, L., Thierauch, K.H. (1999) J Biol chem.274, 5612-doped 5619).

Thus, in another preferred embodiment of the present invention, the antigen or antigenic determinant is a peptide derived from the VEGFR-II contact site. This provides a composition and vaccine composition according to the invention which may have anti-angiogenic properties and which may be useful in cancer therapy. Inhibition of tumor growth in mice with VEGFR-2 specific sera has been demonstrated (Wei, YQ., Wang, QR., Zhao, x., Yang, l., tianan.l., Lu, y., Kang, b., Lu, cj., Huang, MJ., Lou, YY., Xiao, f., He, QM., Shu, JM., Xie, XJ., Mao, YQ., Lei, s., Luo, f., Zhou, LQ., Liu, ce, Zhou, h., Jiang, y., Peng, f., Yuan, LP., Li, q., Wu, y., Liu, JY. (2000) Nature Medicine 6, 1160). Thus, further preferred antigenic determinants suitable for use in the compositions of the present invention and the anti-angiogenic vaccine compositions according to the present invention include human VEGFR-II derived peptides having the sequence CTARTELNVGIDFNWEYPSSKHQHKK, and/or murine VEGFR-II derived peptides having the sequence CTARTELNVGLDFTWHSPPSKSHHKK, and/or the related extracellular globular domains 1-3 of VEGFR-II.

Thus, in a preferred embodiment of the invention, the vaccine composition comprises a core particle selected from a virus-like particle or a bacterial pilus and a VEGFR-II derived peptide or fragment thereof as antigen or antigenic determinant according to the invention.

Those skilled in the medical arts of treating related diseases know how to select antigens or antigenic determinants for compositions and methods of treating other autoantigen-related diseases or conditions. Representative examples of such antigens or antigenic determinants are, for example: lymphotoxins (e.g., lymphotoxin alpha (LT α), lymphotoxin beta (LT β)) and lymphotoxin receptors, receptor activators of nuclear factor kappa B ligand (RANKL), Vascular Endothelial Growth Factor (VEGF), vascular endothelial growth factor receptor (VEGF-R), interleukin-5, interleukin-17, interleukin-13, CCL21, CXCL12, SDF-1, MCP-1, Endoglin, resistin, GHRH, LHRH, TRH, MIF, Eotaxin (Eotaxin), bradykinin, BLC, tumor necrosis factor alpha, and amyloid beta peptide (A β)_1-42) (SEQ ID NO: 220) and fragments thereof that elicit an immune response. In a preferred embodiment, the antigen is an amyloid beta peptide (A β)_1-42) (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) (SEQ ID NO: 220) or a fragment thereof. Amyloid beta protein is SEQ ID NO: 218. amyloid beta precursor protein is SEQ ID NO: 219.

in another preferred embodiment of the invention, the antigen or antigenic determinant is an angiotensin peptide or a fragment thereof. The term "angiotensin" as used herein includes any peptide comprising the sequence of angiotensinogen, angiotensin I or angiotensin II or fragments thereof. The sequence is as follows: angiotensinogen: DRVYIHPFHLVIHN, respectively; angiotensin I: DRVYIHPFHL, respectively; angiotensin II: DRVYIHPF. One or more additional amino acids are typically added at the C-terminus or N-terminus of the angiotensin peptide sequence. The sequence of angiotensin corresponds to the human sequence and is identical to the murine sequence. Thus, immunization of a human or mouse with a vaccine or composition containing such angiotensin peptide as an antigenic determinant in accordance with the present invention, respectively, is vaccination against self-antigens. These other amino acids are particularly valuable for targeted and ordered binding to the core particle.

Preferably, angiotensin peptide has an amino acid sequence selected from the group consisting of: a) amino acid sequence CGGDRVYIHPF; b) amino acid sequence CGGDRVYIHPFHL; e) amino acid sequence DRVYIHPFHLGGC; and d) the amino acid sequence CDRVYIHPFH. Angiotensin I is cleaved from renin (14 amino acids) by the kidney-derived enzyme renin. Angiotensin I is a biologically inactive peptide of 10 amino acids. Angiotensin Converting Enzyme (ACE) further cleaves at the N-terminus to yield biologically active 8 amino acid angiotensin II. The peptides bind to angiotensin receptors AT1I and AT2, resulting in vasoconstriction and aldosterone release.

One vaccine of the present invention comprises at least one angiotensin peptide useful for the treatment of hypertension.

In a particular embodiment of the invention, the antigen or antigenic determinant is selected from the group consisting of: (a) recombinant proteins of HIV, (b) recombinant proteins of influenza virus (e.g., influenza M2 protein or a fragment thereof), (c) recombinant proteins of hepatitis c virus, (d) recombinant proteins of Toxoplasma gondii (Toxoplasma), (e) recombinant proteins of Plasmodium falciparum, (f) recombinant proteins of Plasmodium vivax, (g) recombinant proteins of Plasmodium ovale, (h) recombinant proteins of Plasmodium malariae, (i) recombinant proteins of breast cancer cells, (j) recombinant proteins of kidney cancer cells, (k) recombinant proteins of prostate cancer cells, (l) recombinant proteins of skin cancer cells, (M) recombinant proteins of brain cancer cells, (n) recombinant proteins of leukemia cells, (o) recombinant inhibitory proteins (profiling), (p) recombinant proteins of bee venom nuts, (q) recombinant proteins of allergic allergies, (r) recombinant proteins of food allergy,(s) recombinant proteins of asthma, (t) recombinant proteins of Chlamydia (Chlamydia), and (u) fragments of any of the proteins shown in (a) - (t).

Upon selection of the antigen or antigenic determinant of the composition, at least one second attachment site can be added to the molecule during preparation to create an organized, repetitive array that binds to the non-native molecular scaffold of the invention. One skilled in the art would know what constitutes a suitable second attachment site. Representative examples of second attachment sites include, but are not limited to: an antigen, an antibody or antibody fragment, biotin, avidin, streptavidin, a receptor ligand, a ligand binding protein, an interacting leucine zipper polypeptide, an amino group, a chemical group reactive with an amino group, a carboxyl group, a chemical group reactive with a carboxyl group, a sulfhydryl group, a chemical group reactive with a sulfhydryl group, or a combination thereof.

The association of the first and second attachment sites will depend on the nature of the individual molecules selected, but will comprise at least one non-peptide bond. Depending on the combination of the first and second attachment sites, the nature of this association may be covalent, ionic, hydrophobic, polar, or a combination thereof.

In one embodiment of the invention, the second attachment site may be the FOS leucine zipper protein domain or the JUN leucine zipper protein domain.

In a more specific embodiment of the invention, the second attachment site selected is the FOS leucine zipper protein domain that specifically binds to the JUN leucine zipper protein domain on the non-native molecular scaffold of the invention. The association of JUN with the FOS leucine zipper protein domain provides the basis for the formation of organized, repetitive antigen or antigenic determinant arrays on the surface of the scaffold. The FOS leucine zipper protein domain may be fused to a selected antigen or antigenic determinant at the amino terminus, carboxy terminus, or (if desired) in-frame within the protein.

For illustration, several FOS fusion constructs are listed. Human growth hormone (example 4), bee venom phospholipase A₂(PLA₂) (example 9), ovalbumin (example 10) and HIV gp140 (example 12).

To simplify the generation of FOS fusion constructs, several vectors are disclosed, which provide options for the design and construction of antigens or antigenic determinants (see example 6). Vector pAV1-4 was designed for expression of FOS fusions in E.coli; vectors pAV5 and pAV6 were designed for expression of FOS fusion proteins in eukaryotic cells. The characteristics of these vectors are briefly described below:

1, pAV 1: the vector is designed to secrete a fusion protein containing FOS at the C-terminus into the periplasmic space of E.coli. The gene of interest (g.o.i) can be ligated to the StuI/NotI site of the vector.

pAV 2: the vector is designed to secrete a fusion protein containing FOS at the N-terminus into the periplasmic space of E.coli. The gene of interest (g.o.i) may be ligated to the NotI/EcoRV (or NotI/HindIII) site of the vector.

pAV 3: this vector is designed to produce a fusion protein containing FOS at the C-terminus in the cytoplasm of E.coli. The gene of interest (g.o.i) can be ligated to the EcoRV/NotI site of the vector.

pAV 4: this vector is designed to produce fusion proteins containing FOS at the N-terminus in the cytoplasm of E.coli. The gene of interest (g.o.i) may be ligated to the NotI/EcoRV (or NotI/HindIII) site of the vector. The N-terminal methionine residue was proteolytically removed after protein synthesis (Hirel et al, Proc. Natl. Acad. Sci. USA86: 8247-8251 (1989)).

5, pAV 5: the vector is designed for eukaryotic production of fusion proteins with FOS at the C-terminus. The gene of interest (g.o.i) can be inserted between the sequence encoding the hGH signal sequence and the sequence encoding the FOS domain by ligation into the Eco47III/NotI site of the vector. Alternatively, a gene containing its own signal sequence may be fused to the FOS coding region by ligation into the StuI/NotI site.

pAV 6: the vector is designed for eukaryotic production of fusion proteins with FOS at the N-terminus. The gene of interest (g.o.i) may be ligated to the NotI/StuI (or NotI/HindIII) site of the vector.

One skilled in the art will appreciate that construction of a FOS-antigen or FOS-epitope fusion protein may include the addition of specific genetic elements to facilitate production of a recombinant protein. Example 4 provides guidance for the addition of specific e.coli translational regulatory elements and example 7 provides guidance for the addition of eukaryotic signal sequences. Other genetic elements may also be selected according to the particular needs of the skilled person.

The invention also encompasses the production of FOS-antigen or FOS-epitope fusion proteins in bacteria (example 5) or eukaryotic cells (example 8). One skilled in the art will know which cell type to select for expression of the fusion protein, depending on a variety of factors, such as whether post-translational modification is an important consideration in the design of the composition.

As described previously, the present invention discloses various methods for constructing FOS-antigens or FOS-epitope fusion proteins using pAV vectors. In addition to allowing prokaryotic and eukaryotic expression, these vectors enable the skilled artisan to select whether to add the FOS leucine zipper protein domain to the N-terminus or the C-terminus of the antigen. Specific examples are provided in which N-terminal and C-terminal FOS fusions are formed with PLA2 (example 9) and ovalbumin (example 10). Example 11 demonstrates PLA ₂And purification of ovalbumin FOS fusion protein.

In a more specific embodiment, the invention relates to an antigen or antigenic determinant encoded by the HIV genome. More specifically, this HIV antigen or antigenic determinant is gp 140. As described in examples 11-15, HIV gp140 can be prepared in a form with the FOS leucine zipper protein domain, and the fusion protein synthesized and purified for attachment to the non-natural molecular scaffold of the present invention. One skilled in the art will appreciate that other HIV antigens or antigenic determinants may also be used in the production of the compositions of the present invention.

In another more specific embodiment, the present invention relates to a vaccine composition comprising at least one influenza virus nucleic acid-encoded antigen or antigenic determinant, and the use of such a vaccine composition in eliciting an immune response. In a more specific embodiment, the influenza antigen or antigenic determinant may be an M2 protein (e.g., M2 protein having the amino acid sequence shown in SEQ ID NO: 213, GenBank accession No. P06821, or SEQ ID NO: 212, PIR accession No. MFIV62), or a fragment thereof (e.g., the amino acid sequence of about 2-24 amino acids in SEQ ID NO: 213, SEQ ID NO: 212). In addition, influenza antigens or antigenic determinants may also be coupled to the non-natural molecular scaffold or core particle by peptide or non-peptide bonds. When an influenza antigen or antigenic determinant is coupled to an unnatural molecular scaffold or core particle by a peptide bond, an ordered, repetitive array of molecules in the form of a fusion protein expression product will typically be formed. However, a more preferred embodiment is a composition wherein the M2 peptide is coupled to the Q β capsid protein, HBcAg capsid protein, or pilus disclosed herein by chemical cross-linking.

Different portions of the M2 protein suitable for use in the invention (e.g., the M2 protein having the amino acid sequence of SEQ ID NO: 213), as well as other proteins for which an immune response is sought, may comprise or consist of peptides of any number of amino acids in length, but typically at least 6 amino acids in length (e.g., peptides of 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 97 amino acids in length).

In another embodiment of the invention, the selected second attachment site is a cysteine residue that specifically binds to a lysine residue of the non-natural molecular scaffold or core particle of the invention, or the selected second attachment site is a lysine residue that specifically binds to a cysteine residue of the non-natural molecular scaffold or core particle of the invention. Chemical attachment of lysine residues (Lys) to cysteine residues (Cys) provides the basis for the formation of organized, repetitive antigens or antigenic determinant arrays on the surface of scaffolds or core particles. Cysteine or lysine residues may be engineered in-frame at the amino terminus, carboxy terminus, or (where desired) within the protein of choice for the antigen or antigenic determinant. For example, is PLA ₂And HIV gp140 provides a cysteine residue for attachment to the first attachment site for a lysine residue. In other embodiments, the present invention provides vaccine compositions suitable for use in methods of preventing and/or attenuating allergy (e.g., allergy leading to allergic reactions). Thus, vaccine compositions of the invention include compositions that result in the production of antibodies that can prevent and/or attenuate allergy. Thus, in certain embodiments, the vaccine compositions of the present invention compriseCompositions for eliciting an immune response against an allergen are included. Examples of such allergens include phospholipase A enzymes such as Apismellifera (SEQ ID NO: 168, GenBank accession number 443189; SEQ ID NO: 169, GenBank accession number 229378), Apis dordata (SEQ ID NO: 170, GenBank accession number B59055), Apis cerana (SEQ ID NO: 171, GenBank accession number A59055), Bombus pennyyanius (SEQ ID NO: 172, GenBank accession number B5638) and Heloderma supectorum (SEQ ID NO: 173, GenBank accession number P80003; SEQ ID NO: 174, GenBank accession number S14764; SEQ ID NO: 175, GenBank accession number 226711)₂(PLA₂) A protein.

PLA as Apis mellifera ₂The amino acid sequence of the protein (SEQ ID NO: 168) is an example, and represents intact PLA may also be used in a composition for preventing and/or attenuating allergy₂A peptide of at least about 60 amino acids in length for any portion of the sequence. Examples of such peptides include peptides comprising seq id NO: 168, amino acids 1-60 of SEQ ID NO: 168, amino acids 1-70 of SEQ ID NO: 168, amino acids 10-70 of SEQ ID NO: 168, amino acids 20-80 of SEQ ID NO: 168, amino acids 30-90 of SEQ ID NO: 168, amino acids 40-100 of SEQ ID NO: 168, amino acids 47-99 of SEQ ID NO: 168, amino acids 50-110 of SEQ ID NO: 168, amino acids 60-120 of SEQ ID NO: 168 or amino acids 70-130 of SEQ ID NO: 168, amino acid 90-134, and other PLA₂Proteins (e.g., PLA as described above)₂Protein). Other examples of such peptides include peptides comprising SEQ ID NO: 168, amino acids 1-10 of SEQ ID NO: 168, amino acids 5-15 of SEQ ID NO: 168, amino acid 10-20 of SEQ ID NO: 168, amino acids 20-30 of SEQ ID NO: 168, amino acids 30-40 of SEQ ID NO: 168, amino acids 40-50 of SEQ ID NO: 168, amino acids 50-60 of SEQ ID NO: 168, amino acids 60-70 of SEQ ID NO: 168, amino acids 70-80 of SEQ ID NO: 168, amino acids 80-90 of SEQ ID NO: 168, amino acids 90-100 of SEQ ID NO: 168, amino acid 100-110 of SEQ ID NO: 168 or amino acid 110-120 of SEQ ID NO: 168 amino acid 120-130 peptides, and other PLA ₂Proteins (e.g., PLA as described above)₂Protein).

PLA suitable for use in the present invention₂Portions, as well as portions of other proteins for which an immune response is sought, may comprise or consist of peptides that are typically at least 6 amino acids in length (e.g., peptides that are 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75.80, 85, 90, 95, or 100 amino acids in length).

PLA₂Peptides (e.g., full-length PLA as described above)₂Proteins and portions thereof) can also be coupled to any substance capable of forming an ordered and repetitive antigen array (e.g., Q β capsid protein or fragments thereof).

In another aspect of the invention, the invention provides compositions particularly suited for the treatment and/or prevention of diseases caused or exacerbated by "self" gene products.

In a preferred embodiment of the invention, the antigenic determinant is RANKL (receptor activator of NF κ B ligands). RANKL is also known as TRANCE (TNF-related activation-induced cytokine), ODF (osteoclast differentiation factor), or OPGL (Osteoprotegerin ligand). The amino acid sequence of the extracellular portion of human RANKL is shown in SEQ ID NO: 221(RNAKL _ human: TrEMBL: O14788), while the amino acid sequence of one human isoform is shown in SEQ ID NO: 222. the sequences of the extracellular portions of murine RNAKL and one isoform are shown in SEQ ID NO: 223(RANKL _ mouse: TrEMBL: O35235), SEQ ID NO: 224(RANKL _ mouse spliced form: TrEMBL: Q9JJK8 and TrEMBL: Q9JJK 9).

RANKL has been shown to be an important factor in osteoclastogenesis. Inhibiting the interaction of RANKL with its receptor RNAK inhibits osteoclastogenesis, thereby providing a means to prevent excessive bone resorption that can be seen in osteoporosis and other diseases. The RANKL/RANK interaction is inhibited by a RANK-Fc fusion protein or a soluble decoy receptor for RANKL (called osteoprotegerin OPG).

In the immune system, RANKL is expressed on T cells, while RANK is found on antigen presenting cells. The RANKL-RANK interaction is important for CD 40L-independent T helper cell activation (Bachmann et al, J.exp.Med.7: 1025(1999)) and extends dendritic cell lifespan and enhances its adjuvant properties (Josien et al, J Exp Med.191: 495 (2000)).

In bone, RANKL is expressed on stromal or osteoblasts, while RANK is expressed on osteoclast precursors. The interaction of RANK with RANKL is critical for the development of osteoclast precursors into mature osteoclasts. osteoprotegerin blocks this interaction.

OPG-deficient mice suffer from osteoporosis, which can be rescued by injection of recombinant OPG. This means that OPG is able to reverse osteoporosis. Thus, inhibition of the RANK-RANKL interaction by injection of the composition of this particular embodiment of the invention can reverse osteoporosis.

In addition, arterial calcification was also observed in OPG knockout mice, and OPG injection was able to reverse (Min et al, j.exp.med.4: 463 (2000)). On an adjuvant-induced arthritis model, injection of OPG can prevent bone loss and cartilage destruction, but not inflammation (paw swelling). It is postulated that activated T cells result in RANKL-mediated increased osteoclast production and bone loss. OPG inhibits osteoclastogenesis induced by prostate cancer and prevents the growth of prostate cancer in mouse bone. In mice, OPG can relieve pain in advanced bone cancer.

RANKL is a 245 amino acid transmembrane protein belonging to the TNF superfamily. TACE-like proteases are able to release their extracellular domain part (178 amino acids) (Lum et al, J biol chem.274: 13613 (1999)). In addition, splice variants lacking a transmembrane domain have also been described (lkeda et al, Endocrinology 142: 1419 (2001)). The released fraction contains domains that are highly homologous to soluble TNF-alpha. This extracellular domain of RANKL forms a homotrimer, which can be found in TNF-a. Its C-terminus appears to be associated with the trimer contact site. The sequence region contains a cysteine.

We have modeled the three-dimensional structure of the RANKL counterpart and found that the naturally occurring cysteines may not be accessible in the folded structure interacting with the first attachment site on the vector of the invention. Preferably, a second attachment site comprising an amino acid linker, containing another cysteine residue, is attached to the N-terminus. A human-RANKL construct is a very preferred embodiment of the invention, wherein an amino acid linker comprising a cysteine residue is fused to the extracellular portion of RANKL. However, the fusion of an amino acid linker containing one cysteine residue as second attachment site to the RANKL sequence or the C-terminus of the extracellular portion of RANKL constitutes a further preferred embodiment of the invention.

human-RANKL construct as set forth in SEQ ID NO: 320, prepared according to the disclosure of example 6, one skilled in the art can compare the murine and human RANKL sequences by protein sequence alignment to identify the portion of the human-RANKL sequence that will be cloned into the vector described in example 6. The fragment containing amino acids 138-317 and corresponding to the C-terminal region of the extracellular domain of human RANKL is a particularly preferred embodiment of the invention and may be modified for coupling to VLPs and fimbriae as required by the present invention. However, other suitable vectors may be used for expression in suitable hosts as described below. Also included within the scope of the invention are other human-RANKL constructs, particularly those comprising an extracellular region portion (178 amino acids) or fragment thereof (Lum et al, J Biol chem.274: 13613(1999)) capable of being released by TACE-like proteases, or those comprising sequences corresponding to other splice variants lacking transmembrane domains and conserved fragments thereof. The human C-terminal fragment comprising amino acids 165-317 is also an embodiment of the present invention. Alternatively, fragments comprising the entire extracellular region (amino acids 71-317) which may be modified to couple to VLPs and fimbriae as claimed in the present invention are also included within the scope of the present invention.

RANKL has been expressed in different systems (e.coli, insect cells, mammalian cells) and has been shown to be active, therefore, a variety of expression systems can be employed to produce antigens for compositions. In case the expression of the protein is directed to the periplasm of e.coli, the signal peptide of RANKL or of the RANKL construct consisting of the extracellular part of the protein is replaced by a bacterial signal peptide, both proteins possibly being modified so as to comprise a second attachment site according to the invention. For expression of proteins in the E.coli cytoplasm, the RANKL construct should be free of signal peptide.

In another preferred embodiment of the invention, the antigenic determinant is MIF or a fragment thereof. MIF is a cytokine, the function of which as an inhibitor of macrophage migration was first described in 1966. It is also known as delayed early response protein 6(DER6), glycosylation inhibiting factor or phenylpyruvate tautomerase. The last name arises from the enzymatic activity of MIF, however its endogenous substrate has not been determined.

MIF has been shown to be associated with a variety of diseases. MIF (mRNA and protein) is up-regulated in tuberculin-induced delayed hypersensitivity (DTH) reactions, which are inhibited by anti-MIF antibodies. MIF is also upregulated in allogeneic kidney transplant rejection. In a model of ocular autoimmune disease, experimental autoimmune uveal retinitis (EAU), anti-MIF treatment delayed the development of EAU. MIF is elevated in patient serum, as is in patients with Behcet and iridocyclitis. MIF immunization may be a method of treatment of rheumatoid arthritis.

High serum MIF concentrations are found in atopic dermatitis patients. In skin lesions, MIF is widely expressed, rather than found in the basal cell layer as in the control. MIF concentrations decrease after steroid treatment, which is consistent with the role of MIF in inflammation. MIF has also been found to have an effect on the development of glomerulonephritis. Animals treated with anti-MIF antibodies showed significant relief from glomerulonephritis. MIF is pituitary-produced, secreted, e.g. following LPS stimulation, and exacerbates endotoxemia. Thus, anti-MIF monoclonal antibodies inhibit endotoxemia and septic shock, while recombinant MIF significantly increases lethality in peritonitis. MIF is also a glucocorticoid-induced cytokine production modulator and contributes to inflammation.

MIF is produced by T cells (Th2), supports T cell proliferation, and anti-MIF treatment reduces T cell proliferation and IgG levels. MIF concentrations are elevated in cerebrospinal fluid in patients with multiple sclerosis and neurological Behcet. High MIF levels are also found in the serum of patients with chronic psoriasis. High MIF levels are also found in the serum of patients with ulcerative colitis, but not in patients with crohn's disease.

High MIF levels are found in the serum of patients with bronchial asthma. MIF is also upregulated in synovial fluid of patients with rheumatoid arthritis. anti-MIF treatment is effective in alleviating rheumatoid Arthritis in mouse and rat models (Mikullowska et al, J.Immunol.158: 5514-7 (1997); Leech et al, Arthritis Rheum.41: 910-7 (1998); Leech et al, Arthritis Rheum.43: 827-33 (2000); Santos et al, Clin.exp. Immunol.123: 309-14 (2001)). Thus, treatments aimed at inhibiting MIF activity using compositions comprising MIF as an antigenic determinant may be beneficial for the above mentioned diseases.

MIF from mouse, rat and human consists of 114 amino acids, containing three conserved cysteines, as shown in SEQ ID NO: 225(MIF _ rat: SwissProt), SEQ ID NO: 226(MIF _ mouse: SwissProt) and SEQ ID NO: 227(MIF _ human: SwissProt). The three subunits constitute a homotrimer which is not stabilized by disulfide bonds. The X-ray structure has been resolved and shows three free cysteines (Sun et al, PNAS 93: 5191-96(1996)) and some literature data suggest the presence of disulfide bonds. However, no cysteine is exposed enough to optimally interact with a possible first attachment site on the carrier. Thus, since the C-terminus of the protein is exposed in the trimeric structure, in order to create the second attachment site in a preferred embodiment of the invention, it is preferred to add an amino acid linker containing a free cysteine residue at the C-terminus of the protein, as described in example 4 for rat-MIF.

There is only one amino acid change between mouse-MIF and rat-MIF, and similarly there is very high sequence homology (about 90% sequence identity) between human-MIF and rat-MIF or between human-MIF and mouse-MIF. human-MIF and mouse-MIF constructs according to the invention are described in example 4 and can be produced as disclosed. To confirm that the MIF protein or fragment thereof bound to the core particle of the invention has high potency for inducing self-specific immune responses, mice were injected with a rat-MIF construct coupled to Q β capsid protein. Immunization of mice with rat-MIF resulted in high antibody titers, indicating that immunization with MIF constructs coupled to virus-like particles, in particular to Q β capsid protein, overcome tolerance to autoantigen immunization (example 4). Thus, the compositions of the invention comprising human-MIF protein bound to a core particle, preferably to a pilus or virus-like particle, more preferably to a virus-like particle of an RNA-bacteriophage, even more preferably to RNA-bacteriophage Q β or fr, are highly preferred embodiments of the invention.

However, the addition of an amino acid linker containing a free cysteine at the N-terminus of the MIF sequence leads to a further preferred embodiment of the invention. MIF has been expressed in E.coli, purified, and shown to have full functionality (Bernhagen et al, Biochemistry 33: 14144-. Thus, for the production of a preferred embodiment of the invention, MIF may preferably be expressed in e.

If the starting methionine is not cleaved from the construct, the tautomerase activity of MIF is inhibited. The MIF construct described in example 4, expressed in e.coli, shows tautomerase activity. MIF mutants that have had the initial methionine cleaved and the proline residue in the sequence after the initial methionine mutated to alanine do not exhibit tautomerase activity are other embodiments of the present invention and are included within the scope of the present invention. In certain particular embodiments, the MIF mutant is immunized with no tautomerase activity.

In another preferred embodiment of the invention, the antigenic determinant is interleukin-17 (IL-17). Human IL-17 is a 32kDa, disulfide-linked homodimeric protein with variable glycosylation (Yao, Z et al, J.Immunol.155: 5483-S5486 (1995); Fossiez, F. et al, J.exp. Med.183: 2593-S2603 (1996)). The protein comprises 155 amino acids and an N-terminal secretory signal sequence of 19-23 residues. The amino acid sequence of IL-17 is only similar to the herpes virus protein (HSV13) and has no similarity to other cytokines or known proteins. The amino acid sequence of human IL-17 is set forth in SEQ ID NO: 228 (accession No. AAC50341) shows that the mouse protein sequence is shown in SEQ ID NO: 229 (accession number AAA 37490). In a number of tissues and cell lines evaluated, mRNA transcripts encoding IL-17 were detected only in activated T cells and peripheral blood mononuclear cells stimulated with phorbol 12-myristate 13-acetate/ionomycin (Yao, Z. et al, J.Immunol.155: 5483-5486 (1995); Fossiez, F. et al, J.exp.Med.183: 2593-2603 (1996)). Both human and mouse sequences contain 6 cysteine residues.

The IL-17 receptor is widely expressed in many tissues and cell types (Yao, Z. et al, Cytokine 9: 794-800 (1997)). Although a protein containing a transmembrane domain and a 525 amino acid long intracellular domain can be predicted based on the amino acid sequence (866 amino acids) of the human IL-17 receptor, the receptor sequence is unique and has no similarity to the sequence of any receptor from the cytokine/growth factor receptor family. The fact that IL-17 itself lacks similarity to other known proteins, taken together, suggests that IL-17 and its receptor may be part of a new family of signaling proteins and receptors. Clinical studies have shown that IL-17 may be involved in a variety of inflammatory diseases. IL-17 is secreted by synovial T cells of patients with rheumatoid Arthritis and stimulates the production of inflammatory mediators (Chabaud, M. et al, J.Immunol.161: 409-. High levels of IL-17 have been reported in patients with rheumatoid arthritis (Ziolkowska et al, J.Immunol.164: 2832-8 (2000)).

Interleukin 17 has been shown to have an effect on proteoglycan degradation in the knee joint in mice (DudlerJ. et al, Ann Rheum Dis.59: 529-32(2000)) and to contribute to synovial matrix destruction (Chabaud M. et al, cytokine.12: 1092-9 (2000)). There are related animal arthritis models to test the efficacy of MIF immunization (Chabaud M. et al, cytokine. 12: 1092-9 (2000)). Increased IL-17mRNA levels were found in monocytes of patients with multiple sclerosis (Matusevicius, D. et al, Mult. Scler.5: 101-104 (1999)). Elevated serum IL-17 levels are also found in patients with systemic Lupus erythematosus (Wong C.K. et al, Lupus 9: 589-93 (2000)). In addition, IL-17mRNA levels were also elevated in T cells isolated from lesional psoriatic skin (Teunissen, M.B. et al, J.Invest.Dermatol.111: 645-649 (1998)).

The relevance of IL-17 to renal transplant rejection has also been demonstrated (Fossiez, F. et al, int. Rev. Immunol.16: 541-51 (1998)). Antonysamy et al (J.Immunol.162: 577-84(1999)) presented evidence of a role for IL-17 in organ allograft rejection, demonstrating that IL-17 promotes functional differentiation of dendritic cell progenitors. Their findings suggest a role for IL-17 in allogeneic T cell proliferation, which may be mediated in part by maturation-inducing effects on DCs. Furthermore, the same group reported (Tang J.L. et al, Transplantation 72: 348-50(2001)) a role for IL-17 in the immunopathological pathogenesis of acute vascular rejection, wherein interleukin-17 antagonism inhibits acute vascular rejection but not chronic vascular rejection. IL-17 appears to have potential as a new target for therapeutic intervention in allogeneic transplantation.

The above findings suggest that IL-17 may play a critical role in the initiation or persistence of the inflammatory response (Jovanovic, D.V. et al, J.Immunol.160: 3513-3521 (1998)).

The anti-IL-17 monoclonal antibody mAb5(Schering-Plough institute) was able to completely inhibit IL-6 production in synovial supernatant from Rheumatoid Arthritis (RA) after induction of IL-17 at 50 ng/ml. An unrelated mAb MX1 had no effect in this assay. mAb5 is a mouse IgG1 obtained after immunization with human rIL-17(r ═ recombinant). In this assay system, mAb5 at a concentration of 1. mu.g/ml was able to completely inhibit IL-6 production (Chabaud M. et al, J.Immunol.161: 409-414 (1998)). Thus, IL-17 immunization provides a method for treating a variety of these diseases.

Thus, in another preferred embodiment of the invention, the composition comprises a linker comprising a second attachment site fused to the C-terminus of recombinant IL-17. In other preferred embodiments of the invention, however, an amino acid linker containing a free cysteine is fused to the N-terminus of the sequence corresponding to the processed protein sequence, or inserted at the N-terminus of the mature form of the protein, C-terminus of the signal peptide. For eukaryotic expression systems, the signal peptide of the IL-17 gene may be replaced with another signal peptide, as described herein for other autoantigens, if desired. For expression in dry bacteria, the signal peptide was replaced with a bacterial signal peptide for soluble expression in the periplasm, or deleted for expression in the cytoplasm. Constructs of human IL-17 lacking a signal peptide preferably contain residues 24-155, 22-155, 21-155 or 20-155. Constructs of mouse IL-17 lacking the signal peptide preferably contain residues 26-158, 25-158, 24-158, or 27-155. Human IL-17 can be expressed in CV1/EBNA cells; recombinant hIL-17 was shown to be secreted in glycosylated and non-glycosylated forms (Yao, Z. et al, J.Immunol.155: 5483-5486 (1995)). IL-17 can also be expressed as a hIL-17/Fc fusion protein, from which the IL-17 protein is then cleaved. IL-17 can also be expressed in Pichia pastoris (Pichia pastoris) (Murphy K.P. et al, Protein Expr purify.12: 208-14 (1998)). Human IL-17 can also be expressed in E.coli. When the expression of IL-17 in E.coli is directed to the periplasm, the signal peptide of IL-17 is replaced with a bacterial signal peptide. For expression of proteins in the cytoplasm of E.coli, the IL-17 construct should be free of signal peptide.

In another preferred embodiment of the invention, the antigenic determinant is interleukin-13 (IL-13). IL-13 is a cytokine secreted by activated T lymphocytes, primarily affecting monocytes, macrophages and B cells. The amino acid sequence of precursor human IL-13 is set forth in SEQ ID NO: 230, the amino acid sequence of the processed human IL-13 is shown in SEQ ID NO: 231, respectively. The first 20 amino acids of the precursor protein correspond to the signal peptide and are not present in the processed protein. Mouse sequences have also been reported, and the processed amino acid sequences are shown in SEQ ID NO: 232 (Brown K.D. et al, J.Immunol.142: 679-687 (1989)). Depending on the expression host, for example for expression and secretion in a eukaryotic host, the IL-13 construct will comprise the sequence of the pre-protein or consist of the mature protein, for example for cytoplasmic expression in E.coli. For expression in the periplasm of E.coli, the signal peptide for IL-13 was replaced with a bacterial signal peptide.

IL-13 is a cytokine produced by T helper 2 cells (similar to IL-4, IL-5), and has recently been shown to be associated with allergic airway responses (asthma). Upregulation of IL-13 and IL-13 receptors has been found in a variety of tumor types (e.g., Hodgkin's lymphoma). Interleukin 13 is secreted by Hodgkin and Lei-Shing cells and stimulates their growth (Kapp U et al, J Exp Med.189: 1939-46 (1999)). Thus, IL-13 immunization provides a method of treating the above-mentioned diseases (e.g., asthma or Hodgkin's lymphoma).

Preferably, the composition comprises an amino acid linker containing a free cysteine residue and fused to the N-terminus or C-terminus of the mature IL-13 sequence to introduce a second attachment site within the protein. In other preferred embodiments, an amino acid linker containing a free cysteine is added to the N-terminus of the mature form of IL-13, since the N-terminus is freely accessible according to the NMR structure of IL-13 (Eisenmesser, E.Z. et al, J.mol.biol.310: 231 (2001)). In a further preferred embodiment, the amino acid linker containing a free cysteine is fused to the N-terminus of the sequence corresponding to the processed protein sequence, or is inserted at the N-terminus of the sequence of the mature form of the protein, C-terminus of the signal peptide. In a further preferred embodiment, an amino acid linker containing a free cysteine is added to the C-terminus of the protein.

IL-13 can be expressed in E.coli (Eisenmesser, E.Z. et al, Proten Expr. Purif.20: 186-95(2000)) or NS-0 cells (eukaryotic cell lines) (Cannon-Carlson S. et al, Protein Expr. Purif.12: 238-48 (1998)). Example 9 describes the constructs of murine IL-13 fused to an amino acid linker containing cysteine residues and the expression of the constructs in bacterial and eukaryotic hosts. Human IL-13 constructs can be prepared as described in example 9, producing the proteins human C-IL-13-F (SEQ ID NO: 330) and human C-IL-13-S (SEQ ID NO: 331) after expression of the fusion protein and cleavage with factor Xa and enterokinase, respectively. The proteins so produced are capable of coupling to VLPs and pili, resulting in preferred embodiments of the invention.

In another embodiment of the invention, the antigenic determinant is interleukin-5 (IL-5). IL-5 is a lineage specific cytokine for eosinophil production (eosinophilopoosis) and plays an important role in diseases associated with elevated eosinophil numbers, such as asthma. The sequences of the precursor and processed human IL-5 are set forth in SEQ ID NO: 233 and SEQ ID NO: 234, the processed mouse amino acid sequence is set forth in SEQ ID NO: 235, respectively.

The biological function of IL-5 has been shown in several studies (CoffmanR.L. et al, Science 245: 308-10 (1989); Kopf et al, Immunity 4: 15-24(1996)) indicating a beneficial effect on the inhibition of IL-5 function in eosinophil-mediated diseases. Inhibition of the action of IL-5 provides a method of treating asthma and other eosinophil-related diseases.

IL-5 is covalently linked by a disulfide bond to form a dimer. A single chain (sc) construct has been reported in which two IL-5 monomers are linked by a peptide linker.

In a preferred embodiment of the invention, a peptide linker containing a free cysteine is added to the N-terminus of the sequence in post-processing form of IL-5. Preferably, a linker containing a free cysteine may also be added to the N-terminus of the sequence in its processed form of scIL-5. In other preferred embodiments, the amino acid linker containing a free cysteine is fused to the N-terminus of the sequence corresponding to the processed protein sequence, or is inserted at the N-terminus of the sequence of the mature form of the protein, C-terminus of the signal peptide.

In another preferred embodiment, the linker containing a free cysteine is fused to the C-terminus of the IL-5 sequence, or to the C-terminus of the scIL-5 sequence.

Various expression systems have been described for IL-5, which may be used to prepare the compositions of the present invention. Proudfoot et al (Biochem J.270: 357-61(1990)) describe a bacterial expression system using E.coli. If IL-5 is expressed in the cytoplasm of E.coli, the IL-5 construct does not contain a signal peptide. To prepare the compositions of the invention, insect cells may also be used to produce IL-5 constructs (Pierrot C. et al, biochem. Biophys. Res. Commun.253: 756-60 (1998)). Likewise, baculovirus expression systems (sf9 cells; IngleyE. et al, Eur. J. biochem. 196: 623-9(1991) and Brown P.M. et al, protein Expr. Purif.6: 63-71(1995)) may also be used, and finally, mammalian expression systems (CHO cells) have been reported and may be used to prepare the compositions of the invention (Kodama S et al, J. biochem. (Tokyo) 110: 693-701 (1991)).

Baculovirus expression systems for mouse IL-5 have also been reported (Mitchell et al, biochem. Soc. Trans.21: 332S (1993); Kunimoto DY et al, Cytokine 3: 224-30(1991)) and mammalian cell expression systems using CHO cells (Kodama S et al, Glycobiology 2: 419-27 (1992)).

Example 10 describes the expression of a murine IL-5 construct, wherein the IL-5 sequence is fused at its N-terminus to an amino acid linker containing a cysteine residue for coupling to VLPs and pili. Human constructs can be prepared as described in example 10, resulting in human C-IL-5-E (SEQ ID NO: 335), human C-IL-5-F (SEQ ID NO: 336) and human C-IL-5-S (SEQ ID NO: 337) proteins suitable for coupling to VLPs and fimbriae to produce the preferred embodiment of the present invention.

In another preferred embodiment of the invention, the antigenic determinant is CCL-21. CCL-21 is a chemokine of the CC subfamily, also known as the inducible small cytokine A21, exodus-2, SLD (secondary lymphocyte cytokine), TCA4 (thymus-derived chemotactic agent 4), or 6 Ckine.

CCL21 inhibits hematopoiesis and stimulates chemotaxis of thymocytes, activated T cells, and dendritic cells, but not B cells, macrophages, or neutrophils. It was shown to have preferential activity on naive T cells. It is also a potent chemoattractant for mesangial cells. CCL21 binds to the chemokine receptors CCR7 and CXCR3 (depending on the species). It triggers the rapid termination of integrin-dependent lymphocyte tumbling under physiological shear and is highly expressed by high endothelial venules.

Murine CCL21 inhibited tumor growth and angiogenesis in a human lung carcinoma SCID mouse model (Arenberg et al, Cancer Immunol.Immunother.49: 587-92(2001)) and a mouse colon carcinoma tumor model (Vicari et al, J.Immunol.165: 1992-Aco2000 (2001)). The angiostatic activity of murine CCL21 was also detected in the rat corneal micro-pocket assay (Soto et al, Proc. Natl. Acad. Sci. USA 95: 8205-10 (1998)).

Chemokine receptors CCR7 and CXCR4 have been shown to be up-regulated in breast cancer cells, with their respective ligands CCL21 and CXCL12 highly expressed in organs that are the primary target of metastasis for breast cancer (Muller et al, Nature 410: 50-6 (2001)). In vitro CCL 21-mediated chemotaxis can be blocked by neutralizing anti-CCL 21 antibodies, as can CXCR 4-mediated chemotaxis by the corresponding antibodies. Thus, CCL21 immunization provides a method for treating metastatic spread of cancer, more specifically breast cancer.

Secreted CCL21 consists of 110 or 111 amino acids in mice and humans, respectively. The respective sequences are set forth in SEQ ID NO: 236 (Swissprot: SY21_ human) and SEQ ID NO: 237 (Swissprot: SY21_ mouse). Unlike other CC cytokines, CCL21 contains two additional cysteines in the C-terminal extension. It is assumed that all cysteines are involved in disulfide bonds.

Constructs and expression systems useful for making compositions of the invention comprising CCL21 antigenic determinants are described below. In the NMR structure of the cognate protein eotaxin, both the N-and C-termini are exposed to the solvent. In certain embodiments, an amino acid linker containing a free cysteine as the second attachment site is added to the C-terminus of the protein. A fusion protein containing alkaline phosphatase (at the C-terminus of CCL 21) was expressed and shown to be functional, indicating that the fusion at the C-terminus of CCL21 is compatible with receptor binding. In other embodiments, the free cysteine-containing amino acid linker is fused to the N-terminus of the sequence corresponding to the processed protein sequence, or inserted N-terminal of the sequence of the mature form of the protein, C-terminal of the signal peptide.

Several expression systems have been described for the production of CCL21 (e.g., Hedrick et al, J Immunol.159: 1589-93 (1997)). For example, it can be expressed in a baculovirus system (Nagira et al, J.biol.chem.272: 19518-24 (1997)).

In a related preferred embodiment, the antigenic determinant is stromal derived factor-1 (SDF-1), now designated CXCL 12. CXCL12 is a chemokine produced by bone marrow stromal cells and was originally identified as a stimulator of pre-B cells.

As described above, it has been shown that chemokine receptors CCR7 and CXCR4 are up-regulated in breast cancer cells, while their respective ligands CCL21 and SDF-1 are highly expressed in organs that are the first targets of breast cancer metastasis (Muller et al, Nature 410: 50-6 (2001)). Chemotaxis mediated by SDF-1/CXCR4 in vitro can be inhibited by neutralizing anti-SDF-1 antibodies and anti-CXCR 4-antibodies.

In a SCID mouse breast cancer metastasis model using the human MDA-MB-231 breast cancer cell line, a significant reduction in lung metastasis was observed when mice were treated with anti-CXCR 4 antibody. In draining lymph nodes, a reduction in metastasis to inguinal and axillary lymph nodes was observed (38% metastasis, while 100% in controls). Accordingly, CXCL12 immunization provides a method for treating metastasis of cancer (particularly breast cancer).

SDF-1/CXCR4 chemokine receptors have been shown to enhance the efficiency of homing more primitive hematopoietic progenitors to the bone marrow. In addition, it was also concluded that CXCR4 and SDF-1 can affect the distribution of chronic lymphocytic leukemia cells. These cells constantly infiltrate the bone marrow of patients, showing that their migration in bone marrow is CXCR4 dependent. Chronic lymphocytic leukemia cells undergo apoptosis unless they are co-cultured with stromal cells. SDF-1 blocking antibodies inhibit this protective effect of stromal cells (Burger et al, Blood 96: 2655-63 (2000)). CXCL12 immunization thus provides a method for treating chronic lymphocytic leukemia.

CXCR4 has been shown to be a co-receptor for HIV entry into T cells. SDF-1 inhibits X4(CXCR4 dependent) HIV strain infection of CD4+ cells (Oberlin et al, Nature 382: 833-5 (1996); Bleul et al, Nature 382: 829-33 (1996); Rusconi et al, anti. ther. 5: 199-204 (2000)). Synthetic peptide analogues of SDF-1 have been shown to be effective in inhibiting HIV-1 entry and infection through the CXCR4 receptor (WO059928A 1). Accordingly, CXCL12 immunization provides a means to block HIV entry into T cells and thus a means to treat AIDS.

SDF-1-CXCR4 interaction has also been reported to play a key role in CD4+ T cell accumulation in the synovium of rheumatoid arthritis (Nanki et al, 2000). SDF-1 immunization thus provides a method for treating rheumatoid arthritis.

It is known that human and murine SDF-1 exist in two forms-SDF-1. alpha. and SDF-1. beta. by differential splicing of one gene. They differ by 4C-terminal amino acids, which are present in SDF-1. beta. (74 amino acids) and absent in SDF-1. alpha. (70 amino acids). Human SDF-1 has the sequence shown in SEQ ID NO: 238 (Swissprot: SDF1_ human), the sequence of murine SDF-1 is shown in SEQ ID NO: 239 (Swissprot: SDF1_ mouse). SDF-1 contains 4 conserved cysteines, forming two intramolecular disulfide bonds. The crystal structure of SDF appears as a non-covalently linked dimer (Dealwis et al, PNAS 95: 6941-46 (1998)). The SDF-1 structure also shows a long N-terminal extension.

The (partial) receptor binding sites on SDF-1 were identified using alanine scanning mutagenesis (Ohnishi et al, J. Interferon Cytokine Res.20: 691-.

Constructs and expression systems suitable for producing the compositions of the invention in connection with SDF-1 are described below. The N-and C-termini of SDF-1 were exposed to a solvent. In particular embodiments, an amino acid linker containing a cysteine as the second attachment site is fused to the C-terminus of the protein sequence, while in other particular embodiments, an amino acid linker containing a cysteine as the second attachment site is fused to the N-terminus of the protein sequence. An amino acid linker containing a free cysteine is fused to the N-terminus of the sequence corresponding to the processed protein sequence, or inserted at the N-terminus of the sequence of the mature form of the protein, C-terminus of the signal peptide. The genes encoding these particular constructs can be cloned into an appropriate expression vector.

Expression of SDF-1 in the chick embryo fibroblast Sendai virus system (Moriya et al, FEBS Lett.425: 105-11(1998)) and in E.coli (Holmes et al, prot. Expr. purif.21: 367-77(2001)) and chemical synthesis of SDF-1 (Dealwis et al, PNAS 95: 6941-46(2001)) have been reported.

In another preferred embodiment of the invention, the antigenic determinant is BLC. B lymphocyte chemoattractants (BLC, CXCL13) are expressed in the spleen, the Pair knot and the lymph nodes (Gunn et al, 1998). It is most strongly expressed in germinal centers, where B cells undergo somatic mutation and affinity maturation. It belongs to the CXC chemokine family, the closest homolog of which is GRO α _ (Gunn et al, Nature 391: 799-803 (1998)). Human BLC is 64% homologous to murine BLC. The receptor is CXCR 5. BLC also has homology to IL-8. BLC recruits B cells to the follicles of secondary lymphoid organs such as the spleen and pell. BLC is also required for B-cell supplementation in the lymph node compartment rich in Follicular Dendritic Cells (FDC) (Ansel et al, Nature 406: 309-314 (2000)). BLC also induces enhanced expression of lymphotoxin α 1 β 2 (LT. This provides a positive feedback loop, since LT? α 1 β 2 promotes BLC expression (Ansel et al, Nature 406: 309-314 (2000)). BLC has also been shown to induce lymphogenesis (Luther et al, Immunity 12: 471-481 (2000)). FDC also appears to express BLC. Thus, BLC immunization provides a means for treating autoimmune diseases associated with lymphoid neogenesis, such as rheumatoid synovitis and rheumatoid arthritis or type I diabetes. A BLC construct carrying a C-terminal his tail has been described which is functional (Ansel, K.M. et al, J.Exp.Med.190: 1123-1134 (1999)).

Thus, in a preferred embodiment of the invention, the composition comprises a linker containing a cysteine residue as the second attachment site and fused at the C-terminus of the BLC sequence.

In IL-8 homologous to BLC, both the N-and C-termini are free. Thus in another preferred embodiment, an amino acid linker containing a cysteine as second attachment site is added to the N-terminus of the BLC, resulting in this particular composition of the invention.

In other preferred embodiments of the invention, the composition comprises a free cysteine-containing amino acid linker fused to the N-terminus of the sequence corresponding to the processed protein sequence, or inserted C-terminal to the signal peptide at the N-terminus of the sequence of the mature form of the protein. The genes encoding these particular constructs can be cloned into appropriate expression vectors and expressed accordingly. The sequence of human BLC is set forth in SEQ ID NO: 240 (accession number: NP 006410). Amino acids 1-22 of the sequence are signal peptides. The murine sequence is set forth in SEQ ID NO: 241 (accession number: NP 061354). Amino acids 1-21 are signal peptides. To produce the compositions of the invention, the compositions of the invention containing BLC as an antigenic determinant preferably employ the mature form of the protein.

In another embodiment, the antigenic determinant is eotaxin. Eotaxin is a chemokine specific for chemokine receptor 3 and is present on eosinophils, basophils and Th2 cells. However, eotaxin appears to be highly eosinophil-specific (Zimmerman et al, J.Immunol.165: 5839-46 (2000)). Eosinophil migration was reduced by 70% in eotaxin-1 knock-out mice, yet it still developed hypereosinophilia (Rothenberg et al, J.Exp.Med.185: 785-90 (1997)). IL-5 appears to be responsible for the migration of eosinophils from bone marrow into the blood, and eotaxin is responsible for local migration in tissues (Humbles et al, J.exp.Med.186: 601-12 (1997)).

The human genome contains 3 eotaxin genes: 1-3 parts of eotaxin. They have 30% homology to each other. Two genes are known so far in mice: eotaxin 1 and eotaxin 2(Zimmerman et al, J.Immunol.165: 5839-46 (2000)). They have a homology of 38%. Murine eotaxin-2 has 59% homology with human eotaxin-2. In mice, eotaxin-1 appears to be ubiquitously expressed in the gastrointestinal tract, while eotaxin-2 appears to be expressed primarily in the jejunum (Zimmerman et al, J.Immunol.165: 5839-46 (2000)). Eosinophil-activated chemokine-1 is present in bronchial-alveolar fluid (Teixeira et al, J.Clin invest.100: 1657-66 (1997)). The sequence of human eotaxin-1 is shown in SEQ ID NO: 242 (amino acids 1-23 correspond to the signal peptide), the sequence of human eotaxin-2 is shown in SEQ ID NO: 243 (amino acids 1-26 correspond to the signal peptide), the sequence of human eotaxin-3 is shown in SEQ ID NO: 244 (amino acids 1-23 correspond to the signal peptide), the sequence of mouse eotaxin-1 is shown in SEQ ID NO: 245 (amino acids 1-23 correspond to the signal peptide), the sequence of mouse eotaxin-2 is shown in SEQ ID NO: 246 (amino acids 1-23 correspond to the signal peptide).

The molecular weight of eotaxin is 8.3 kDa. It equilibrates between monomer and dimer under a variety of conditions, with an estimated Kd of 1.3mM at 37 ℃ (Crump et al, J.biol.chem.273: 22471-9 (1998)). However the monomeric form is predominant. The structure of eotaxin has been elucidated by NMR spectroscopy. The binding site to its receptor CCR3 is located at the N-terminus, and the region preceding the first cysteine is very important (Crump et al, J.biol.chem.273: 22471-9 (1998)). This finding was confirmed by chemokine receptor peptides that bind eotaxin. Eotaxin contains 4 cysteines, forming 2 disulfide bonds. Thus, in a preferred embodiment, the composition of the invention comprises an amino acid linker comprising a cysteine residue as the second attachment site, preferably fused to the C-terminus of the eotaxin sequence. In other preferred embodiments, the amino acid linker containing a free cysteine is fused to the N-terminus of the sequence corresponding to the processed protein sequence, or inserted N-terminal of the sequence of the mature form of the protein, C-terminal of the signal peptide. The genes encoding these particular constructs are cloned into appropriate expression vectors.

Eotaxin can be chemically synthesized (Clark-Lewis et al, Biochemistry 30: 3128-3135 (1991)). Expression of eotaxin-1 in the cytoplasm of E.coli is also described (Crump et al, J.biol.chem.273: 22471-9 (1998)). Expression of eotaxin-2 in E.coli as an inclusion body which is subsequently refolded (Mayer et al, Biochemistry 39: 8382-95(2000)) and insect cell expression (Forssmann et al, J.Exp.Med.185: 2171-6(1997)) has been described and may be used to implement particular embodiments of the invention.

In another embodiment of the invention, the antigenic determinant is macrophage colony stimulating factor (M-CSF or CSF-1). M-CSF or CSF-1 is a regulator of proliferation, differentiation and survival of macrophages and their myeloid progenitors. The receptor for M-CSF is a cell surface tyrosine kinase receptor, encoded by the proto-oncogene cfms. The increased expression of M-CSF and its receptors has been associated with a poor prognosis in several epithelial cancers, such as breast, uterine and ovarian cancers. Transgenic mice susceptible to breast cancer (PyMT) were crossed with mice containing a recessive null mutation in the csf-1 gene to generate a mouse breed, which was used to study tumor progression. These mice showed reduced late invasive cancer and lung metastasis compared to PyMT mice (Lin et al, J.Exp.Med.193: 727-739 (2001)). The reason seems to be the failure to recruit macrophages to the tumor tissue. Subcutaneous growth of Lewis lung carcinoma was also attenuated in csf.1 nude mice. It is postulated that the mechanism by which macrophages enhance tumor growth is by angiogenic factors, growth factors and proteases produced by macrophages.

There is structural data on the soluble form of M-CSF (crystal structure: Pandit et al, Science 258: 1358-62(1992)) indicating that both the N-and C-termini of the protein are accessible. However, the N-terminus is near the portion that interacts with the receptor. In addition, M-CSF exists in both soluble and cell surface forms, with a transmembrane region located at the C-terminus. Thus, in a preferred embodiment of the invention, the composition of the invention comprises an amino acid linker comprising a cysteine, preferably added at the C-terminus of M-CSF or a fragment thereof, or preferably at the C-terminus of a soluble form of M-CSF or a fragment thereof. In another preferred embodiment, the amino acid linker containing a free cysteine is fused to the N-terminus of the sequence corresponding to the sequence of the processed protein or soluble form of the protein, or inserted at the N-terminus of the sequence of the mature form of the protein or soluble form of the protein, C-terminus of the signal peptide. M-CSF is a dimer in which two monomers are linked by an interchain disulfide bond.

An E.coli expression system for the N-terminal 149 amino acid fragment (functional) of M-CSF has been described (Koths et al, mol. reprod. Dev.46: 31-37 (1997)). Such fragments of M-CSF, preferably modified as described above, are a preferred antigenic determinant of the invention.

The human sequence is set forth in SEQ ID NO: 247 (accession number: NP _ 000748). Further preferred antigenic determinants of the invention include antigenic determinants consisting of SEQ ID NO: 247, residues 33-181 or 33-185, which corresponds to the soluble form of the receptor.

Mouse sequence (accession number: NP-031804) the sequence shown in SEQ ID NO: 248, respectively. The mature sequence begins at amino acid 33. Thus, a preferred antigenic determinant of the invention comprises amino acids 33-181 or 33-185.

In another embodiment, the antigenic determinant is resistin (Res). Passive immunization studies were performed with rabbit polyclonal antibodies raised against mouse resistin (mRes) and GST fusion proteins expressed in bacteria. This passive immunization results in enhanced glucose uptake in an animal model of obesity/type II diabetes (Steppan et al, Nature 409: 307-12 (2001)).

Resistin (Res) is a 114 amino acid peptide hormone of approximately 12 KD. It contains 11 cysteines, of which the most N-terminal one is shown to be responsible for protein dimerization, and the other 10 are thought to be involved in intramolecular disulfide bonds (Banerjee and Lazar, J.biol.chem.276: 25970-3 (2001)). Mutation of the first cysteine to alanine disrupts mRes dimerization.

It HAs been shown that in an animal model mRes containing a FLAG tail at the C-terminus still retains activity (Steppan et al, Nature 409: 307-12(2001)), and similarly, the resistin form with an HA tail (hemagglutinin tail) at the C-terminus is active in tissue culture assays (Kim et al, j.biol. chem.276: 11252-6(2001)), suggesting that the C-terminus is not particularly sensitive to the introduction of modifications. Thus, in a preferred embodiment, the composition of the invention comprises an amino acid linker containing a cysteine as the second attachment site, fused to the C-terminus of the resistin sequence. In another preferred embodiment, an amino acid linker containing a free cysteine is fused to the N-terminus of the sequence corresponding to the processed protein sequence, or inserted at the N-terminus of the mature form of the protein, C-terminus of the signal peptide.

As a preferred embodiment of the invention, MRes or huRes may also be expressed in eukaryotic expression systems as Fc fusion molecules containing a protease cleavage site between resistin and the Fc portion of the construct, preferably C-terminal to one or more cysteine residues in the hinge region of the Fc portion of the fusion protein, or more preferably expressed as described and disclosed in example 2. Cleavage of the fusion protein releases the resistin and it may also comprise an amino acid linker containing cysteine residues as described in example 2, or a part or all of the hinge region of the Fc portion of the fusion protein containing cysteine residues at the C-terminus suitable for coupling to VLPs or pili. The human resistin sequence is shown in SEQ ID NO: 249 (accession number: AF 323081). The mouse sequence is set forth in SEQ ID NO: 250 (accession number: AF 323080). A preferred embodiment of the invention is a human resistin protein fused at the C-terminus to an amino acid linker containing a cysteine residue. Human resistin constructs can be prepared as disclosed in example 2 and the portions of human resistin sequences to be cloned into the vectors described in examples 1 and 2 or other suitable expression vectors are determined by comparing the protein sequence alignment of mouse to human resistin sequences. Examples of human resistin constructs suitable for use in producing compositions of the invention are human resistin-C-Xa (SEQ ID NO: 325), human resistin-C-EK (SEQ ID NO: 326) and human resistin-C (SEQ ID NO: 327)

The human resistin constructs so produced are a preferred embodiment of the invention. Thus, vaccination with the above-described compositions of the present invention provides a method for treating type II diabetes and obesity.

In another embodiment, the antigenic determinant is lymphotoxin- β. Lymphotoxin-beta immunization is useful for treating prion-mediated diseases. Replication of the causative agent of scrapie, a prion-mediated disease, is thought to occur primarily in lymphoid tissues and is dependent on Follicular Dendritic Cells (FDCs) expressing prion-proteins (Brown et al, Nature Med. 11: 1308, 1312 (1999)). It was subsequently shown that mice lacking functional follicular dendritic cells showed reduced prion replication in the spleen and (slightly) delayed nerve invasion (Montrasio et al, Science 288: 1257-. This can be achieved by injecting mice with soluble lymphotoxin- β receptor-Fc-fusion protein (LT β R-Fc). This soluble receptor construct inhibits the development of FDC by interfering with the interaction of T, B or lymphotoxin- β on NK cells with lymphotoxin- β receptors on FDC precursor cells. Thus, lymphotoxin-beta (also known as TNF γ) vaccination can provide a vaccine to treat or prevent Creutzfeldt-Jakob disease (variant) or other prion-mediated diseases, thereby preventing prion replication and neuro-invasion.

Lymphotoxin-beta immunization may also provide a method for treating diabetes. Transgene expression of soluble LT β R-Fc fusion proteins in nonobese diabetic NOD mice prevented diabetes development, but not insulitis (Ettinger et al, J.Exp.Med.193: 1333-40K (2001)). Wu et al (J.Exp.Med.193: 1327-32(2001)) also used NOD mice to study the effect of lymphotoxin-beta, but they did not use transgenic animals, but injected with LT beta R-Fc fusion proteins. They observed strong inhibition of diabetes development and inhibition of insulitis. Most interestingly, treatment with the fusion protein even reversed already existing insulitis. The formation of lymphoid follicular structures in the pancreas can then be reversed. Thus, lymphotoxin- β vaccination provides a method for treating type I diabetes.

The sequence of the human lymphotoxin-beta ectodomain is shown in SEQ ID NO: 250 (TNFC _ human), the sequence of the murine lymphotoxin- β ectodomain is shown in SEQ ID NO: 251 (TNFR _ mouse).

In yet another preferred embodiment, the composition of the invention comprises an amino acid linker containing a free cysteine added to the N-terminus of the sequence corresponding to the processed form of lymphotoxin- β, or inserted between the N-terminus of the sequence corresponding to the mature form of the protein and the signal peptide, or at the C-terminal side of the signal peptide. In a further preferred embodiment of the invention, the extracellular part of lymphotoxin- β is expressed as a fusion protein comprising glutathione-S-transferase fused at the N-terminus to lymphotoxin- β, or a 6 histidine tail fused at the N-terminus to the extracellular part of lymphotoxin- β, followed by a myc-tail. An amino acid spacer containing a protease cleavage site and a linker sequence containing a free cysteine as a second attachment site at the C-terminus of the protease cleavage site are fused to the N-terminus of the lymphotoxin- β extracellular portion sequence. Preferably, the extracellular portion of lymphotoxin- β consists of a fragment corresponding to lymphotoxin- β amino acids 49-306 or 126-306. These particular compositions of the invention can be cloned and expressed in pCEP-Pu eukaryotic vectors. In another preferred embodiment, the composition of the invention comprises an amino acid linker comprising a free cysteine residue suitable as a second attachment site, the linker being fused to the C-terminus of lymphotoxin- β or a fragment of lymphotoxin- β. At one is In a particularly preferred embodiment, the amino acid sequence LACGG is fused N-terminally to the lymphotoxin- β extracellular portion or to a fragment of the lymphotoxin- β extracellular portion, the amino acid sequence LACGG comprising the amino acid linker ACGG which itself comprises a cysteine residue which is coupled to VLPS and to pili, and the protein human C-LT. cndot.after cleavage of the corresponding fusion protein expressed in the vector pCEP-SP-GST-EK or the vector pCP-SP-his-myc-EK as described in example 3 with enterokinase results in the protein human C-LT. cndot._49-306(SEQ ID NO: 346) and human C-LT_126-306(SEQ ID NO：347)。

In a preferred embodiment, the antigen or antigenic determinant is a prion protein, a fragment thereof, in particular a peptide of a prion protein. In one embodiment, the prion protein is a human prion protein. Guidance on how to modify human prion protein for binding to the core particle is provided throughout the specification, particularly in example 7. Mouse prion protein constructs have been disclosed, human prion protein constructs can also be established, and contain, for example, SEQ ID NO: 348, respectively. Other constructs comprise the complete sequence of human prion protein or other fragments of human prion protein, which are other compositions of the invention. Prion protein immunization can provide a means for treating or preventing creutzfeldt-jakob disease (variant) or other prion protein-induced diseases. Immunization with a composition of the invention comprising a prion protein provides a method for treating prion-mediated diseases in other animals, and bovine and ovine prion protein constructs having the respective sequences set forth in SEQ id nos: 349 and SEQ ID NO: 350. The peptide of the human prion protein, which corresponds to the murine peptide described in example 8, and the peptides of amino acid sequences CSAMSRPIIHFGSDYEDRYYRENMHR ("human cprplong") and CGSDYEDRYYRENMHR ("human cprpshrt") are preferred embodiments of the invention. These peptides comprise an N-terminal cysteine residue added for coupling to VLPs and pili. The corresponding bovine and ovine peptides are CSAMSRPLIHFGNDYEDRYYRENMHR ("bovine cprpong") and CGNDYEDRYYRENMHR ("bovine cprpshrt"), CSAMSRPLIHFGNDYEDRYYRENMYR ("ovine cprpong") and CGNDYEDRYYRENMYR ("ovine cprpshrt"), both embodiments of the present invention.

In another preferred embodiment of the invention, the antigenic determinant is tumor necrosis factor alpha (TNF-alpha), a fragment thereof or a peptide of TNF-alpha. In particular, peptides or fragments of TNF-alpha can be used to induce an auto-specific immune response against the entire protein by immunizing a human or animal with vaccines and compositions comprising these peptides or fragments of the invention, respectively. Preferably, with VLPs, phages or bacterial pili as core particles, TNF- α, a peptide or fragment thereof is attached thereto according to the invention.

The following murine peptides are murine homologues of human peptides which have been shown to be bound by antibodies that neutralize TNF- α activity (Yone et al, J.biol.chem.270: 19509-19515) and, in another preferred embodiment of the invention, are modified with cysteine residues for coupling to VLPs, bacteriophages or bacterial pili.

MuTNF- α peptide: the sequence CGG was added to the N-terminus of an epitope consisting of amino acid residues 22-32 of mature murine TNF- α: CGGVEEQLEWLSQR are provided.

3' TNF II peptide: the sequence CGG is fused at the C-terminal of an epitope consisting of amino acid residues 4-22 of mature mouse TNF-alpha, and glutamine at position 21 is mutated into glycine. The sequence of the resulting peptide was: SSQNSSDKPVAHVVANHGVGGC are provided.

5' TNF II peptide: one cysteine residue is fused with the N end of the epitope consisting of amino acid residues 4-22 of mature mouse TNF-alpha, and 21-bit glutamine is mutated into glycine. The sequence of the resulting peptide was: CSSQNSSDKPVAHVVANHGV are provided.

The human sequence corresponding to epitope 4-22 is SSRTPSDKPVAHVVANPQAEGQ. As with the murine sequences, for covalent coupling to VLPs, phages or bacterial pili according to the invention, a cysteine is preferably fused at the N-terminus of the epitope, or the sequence GGC is fused at the C-terminus of the epitope. However, it is within the scope of the invention to include other cysteine-containing sequences fused at the N-or C-terminus of the epitope. In general, it is preferred to insert one or two glycine residues between the added cysteine residue and the epitope sequence. However, instead of glycine residues, other amino acids may be inserted, these amino acid residues preferably being small amino acids, such as serine.

The human sequence corresponding to amino acid residues 22-32 is QLQWLNRRANA. Preferably, for covalent coupling to a VLP or bacterial pilus according to the invention, the sequence CGG is fused at the N-terminus of the epitope. Other TNF- α epitopes suitable for use in the present invention have also been described and disclosed, for example, by Yone et al (J.biol.chem.270: 19509-19515).

The invention also includes compositions comprising mimotopes of the antigens or antigenic determinants described herein.

This particular composition of the invention comprises an antibody or (preferably) an antibody fragment presented on a virus-like particle or a pilus to induce an immune response against the antibody. To elicit a protective immune response against lymphoma, antibodies or antibody fragments produced by the lymphoma cells may be selected, attached to and immunized with the virus-like particle.

In other embodiments, antibodies or antibody fragments that mimic an antigen are attached to the particles. Such a mimobody or antibody fragment may be produced as follows: immunization, followed by isolation of the mimobody or antibody fragment, is carried out by any known method known in the art, including, for example: hybridoma technology (Gherardi, E.et al, J.Immunol. Methods 126: 61-68(1990)), phage display (Harrison ET al, Methods enzymol. 267: 83-109(1996)), ribosome display (Hanes, J.et al, Nat.Biotechnol. 18: 1287-1292(2000)), yeast two-hybrid (Visintin, M.et al, Proc.Natl.Acad.Sci.USA 96: 11723-11728(1999)), yeast surface display (Boder, ET. & Wittrup, KD.methods. enzym.328: 430-444(2000)), bacterial surface display (Daugherty, PS. ET al, Protein Eng.12: 613- (1999)). The mimobodies may also be isolated from the antibody library or the primary antibody library by methods well known in the art, such as those described above.

In another embodiment, an antibody that recognizes the binding site of another antibody, i.e., an anti-idiotype antibody, also known as an immunological antibody, may be used. Antibodies that can be recognized by anti-idiotype antibodies are also referred to as neutralizing antibodies. Thus, by immunization with anti-idiotype antibodies, molecules with neutralizing antibody specificity are produced in situ; we refer to these antibodies produced as induced antibodies. In another preferred embodiment, the immune antibody is selected to interact with a ligand molecule of an immune target molecule. The ligand molecule may be any molecule that interacts with the target molecule, but preferentially interacts with a site of the target molecule where it is desired to generate an antibody to inhibit its function. The ligand molecule may be the natural ligand of the target molecule or may be any ligand engineered, designed or isolated with appropriate binding properties.

The immunizing antibody may be of human origin, such as isolated from a primary or immunized human antibody library, or may be isolated from a library of another animal origin (e.g., murine).

The coupling of the antibody or antibody fragment to the VLP or pilus is achieved as follows: a limited reduction of the exposed disulfide bond (e.g., the interchain disulfide bond between CH1 and ck or C λ in a Fab fragment), or a linker containing a free cysteine fused at the C-terminus of the antibody or antibody fragment. In another embodiment, a linker containing one free cysteine residue is fused to the N-terminus of the antibody or antibody fragment for attachment to the VLP or pilin.

Vaccine compositions employing mimotopes are known in the art, as are methods for producing and identifying mimotopes for particular epitopes. For example, Arnon et al, Immunology 101: 555-562(2000) describes a mimotope-based vaccine against Schistosoma mansoni (Schistosoma mansoni), the complete disclosure of which is incorporated herein by reference. The mimotopes employed for these vaccines were obtained as follows: screening the solid-phase 8mer random peptide library to identify the mimotopes of the epitopes recognized by the protective monoclonal antibody against Schistosoma mansoni. Similarly, Olazewska et al, Virology 272: 98-105(2000) describe the identification of synthetic peptides that mimic epitopes of the measles virus fusion protein and the use of these peptides in the immunization of mice, the complete disclosure of which is incorporated herein by reference. In addition, zuecrcher et al, eur.j.immunol.30: 128-135(2000), the complete disclosure of which is incorporated herein by reference, describes compositions and methods for oral anti-IgE immunization using epitope-displaying phages. In particular, the epitope-displaying M13 phage was used as a vehicle for an oral anti-IgE vaccine. The vaccine compositions tested contained the mimotopes and epitopes of the monoclonal anti-IgE antibody BSW 17.

The invention therefore includes vaccine compositions comprising mimotopes that elicit an immune response directed against a particular antigen, as well as specific mimotope/core particle conjugates and specific mimotope/non-natural molecular scaffold conjugates that make up these vaccine compositions, and the use of these vaccine compositions to elicit an immune response directed against a particular antigen or antigenic determinant. The mimotope may also be a polypeptide, such as an anti-idiotypic antibody. Thus, in a further preferred embodiment of the invention, the antigen or antigenic determinant is an anti-idiotypic antibody or anti-idiotypic antibody fragment.

The invention also includes compositions comprising mimotopes of the antigens or antigenic determinants described herein.

Mimotopes of a particular antigen can be generated or identified by a variety of methods, including screening random peptide phage display libraries (see, e.g., PCT publication No. WO97/31948, the entire contents of which are incorporated herein by reference). Screening of these libraries is often performed in order to identify peptides that bind to one or more antibodies with a particular antigen specificity.

Mimotopes suitable for use in the vaccine compositions of the present invention may be linear or cyclic peptides. The mimotopes, which are linear or cyclic peptides, may be linked to the non-natural molecular scaffold or core particle by a bond other than a peptide bond.

As described above, several human IgE mimotopes and epitopes that elicit immune responses against human IgE molecules have been identified (see, e.g., PCT publication No. WO 97/31948). Thus, in certain embodiments, the vaccine compositions of the present invention include compositions that elicit an immune response against immunoglobulin molecules (e.g., IgE molecules).

Peptides that can be used to elicit such an immune response include proteins, protein subunits, domains of the IgE molecule, and mimotopes that induce the production of antibodies specific for the IgE molecule. The IgE molecule portion used to prepare the vaccine composition is typically derived from an IgE molecule of the species to which the composition is to be administered. For example, vaccine compositions intended for administration to humans typically contain one or more portions of a human IgE molecule, and/or one or more mimotopes capable of eliciting an immune response against a human IgE molecule.

In a specific embodiment, the vaccine composition of the invention for use in humans comprises SEQ ID NO: 176; at least a portion of the IgE heavy chain constant region shown under accession number AAB59424(SEQ ID NO: 176). In a more particular embodiment, the IgE peptide used to prepare the vaccine composition of the invention comprises or consists of a peptide having the following amino acid sequence: CGGVNLTWSRASG(SEQID NO：178)。

In other embodiments, the vaccine compositions of the present invention comprise at least one mimotope that elicits an immune response that produces antibodies specific for a particular antigen.

Examples of IgE mimotopes suitable for the preparation of the vaccine compositions of the present invention include peptides having the following amino acid sequences:

analog bit	SEQ IDNO	Analog bit	SEQ IDNO
				INHRGYWVRNHRGYWVRSRSGGYWLWVNLTWSRASGC.H3Epitope VNLPWSRASGVNLTWSFGLEVNLPWSFGLEC.H3Analog bit VNRPWSFGLE	179180181182183184185186	VKLPWRFYQVVWTACGYGRMGTVSTLSLLDSRYWQPAHSLGLWGMQGRLTLSHPHWVLNHFVSSMGPDQTLRVNLTWSGEFCINHRGYWVCGDPA	187188189190191192193194195216

C. Preparation of alpha vaccine particles

The present invention provides novel compositions and methods for constructing ordered, repetitive antigen arrays. One skilled in the art will appreciate that the conditions for assembly of an ordered, repetitive antigen array will depend largely on the choice of the first attachment site for a particular non-natural molecular scaffold and the choice of the second attachment site for a particular antigen or antigenic determinant. Thus, the choice of the skilled person in the composition design (i.e. the choice of the first and second attachment sites, the antigen and the non-natural molecular scaffold) will determine the specific conditions for the assembly of the alpha vaccine particles (the combined ordered and repetitive antigen array and the non-natural molecular scaffold). The skilled artisan is aware of information regarding the assembly of alpha vaccine particles and there is a wealth of reference to assist the skilled artisan (e.g., molecular cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); modern methods of molecular biology, written by Ausubel, F. et al, John H.Wiley & Sons, Inc. (1997); Celis, cell biology, written by J. Acad Press, 2 nd edition (1998); Harlow, E. and Lane, D., antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y. (1988), all incorporated herein by reference).

In a specific embodiment of the invention, the first and second attachment sites of the invention employ the JUN and FOS leucine zipper protein domains, respectively. In the preparation of alpha vaccine particles, antigens must be produced and purified under conditions that promote the assembly of ordered, repetitive antigen arrays on non-native molecular scaffolds. In the JUN/FOS leucine zipper protein domain embodiment, the FOS-antigen or FOS-epitope should be treated with a reducing agent (e.g., dithiothreitol, DTT) to reduce or eliminate the chance of disulfide bond formation (example 15).

To prepare a non-native molecular scaffold of the JUN/FOS leucine zipper protein domain embodiment (i.e., recombinant Sindbis virus), recombinant E2-JUN virus particles should be concentrated, neutralized, and treated with a reducing agent (see example 16).

The assembly of ordered, repetitive antigen arrays in the JUN/FOS embodiment is performed in the presence of redox shuttle (shuffle). E2-JUN virus particles and 240 times molar excess of FOS antigen or FOS antigenic determinants at 4 degrees C binding for 10 hours. The alpha vaccine particles were then concentrated and purified by chromatography (example 16).

In another embodiment of the invention, the coupling of the non-native molecular scaffold to the antigen or antigenic determinant may be achieved by chemical cross-linking. In a particular embodiment, the chemical agent is a heterobifunctional crosslinker, such as N-hydroxysuccinimide ester of epsilon-maleimidocaproic acid (Tanimori et al, J.Pharm. Dyn.4: 812 (1981); Fujiwara et al, J.Immunol. meth.45: 195(1981)) which contains (1) a succinimide group reactive with an amino group, and (2) a maleimide group reactive with an SH group. The heterologous protein or polypeptide of the first attachment site may be constructed to contain one or more lysine residues as reactive sites for the succinimide moiety of the heterobifunctional crosslinker. Once chemically coupled to a lysine residue of a heterologous protein, the maleimide group of the heterobifunctional crosslinker will be available to react with the SH group of a cysteine residue on an antigen or antigenic determinant. In this case, the preparation of the antigen or antigenic determinant may require the construction of a cysteine residue into the protein or polypeptide selected as the second attachment site so that it can react with a free maleimide functional group on the cross-linker that has been bound to the first attachment site of the non-natural molecular scaffold. Thus, in this case, the heterobifunctional cross-linking agent binds to a first attachment site of the non-natural molecular scaffold and links the scaffold to a second attachment site of an antigen or antigenic determinant.

3. Compositions, vaccines and administration thereof, and methods of treatment

The present invention provides vaccine compositions useful for preventing and/or alleviating a disease or condition. The invention also provides vaccination methods for preventing and/or alleviating a disease or symptom in an individual.

In one embodiment, the invention provides vaccines for the control of infectious diseases in a wide range of species (particularly mammalian species, such as humans, monkeys, cows, dogs, cats, horses, pigs, etc.). Vaccines can be designed to treat infections with viral pathogens such as HIV, influenza, herpes, viral hepatitis, EB, polio, viral encephalitis, measles, chickenpox, and the like; or infection by bacterial pathogens such as pneumonia, tuberculosis, syphilis, etc.; or infection by parasitic pathogens such as malaria, trypanosomiasis, leishmaniasis, trichomoniasis, amebiasis, etc.

In another embodiment, the invention provides vaccines for the control of cancer in a wide range of species (particularly mammalian species, such as humans, monkeys, cows, dogs, cats, horses, pigs, etc.). Vaccines can be designed to treat all types of cancer: lymphoma, carcinoma, sarcoma, melanoma, and the like.

In another embodiment of the invention, the compositions of the invention may be used in the design of vaccines for the treatment of allergy. Antibodies of the IgE isotype are important components in allergy. Mast cells bind IgE antibodies on their surface and are specific The antigen releases histamine and other mediators of allergic reactions upon binding to IgE molecules bound to the surface of mast cells. Therefore, inhibition of IgE antibody production is a promising target for protection from allergy. This will be possible after the desired T helper cell response is obtained. T helper cell responses can be classified as type 1 (T)_H1) And type 2 (T)_H2) T helper cell response (Romagnani, immunol. today18: 263-266(1997)). T is_H1 cells secrete interferon-gamma and other cytokines that trigger B cells to produce IgG1-3 antibodies. In contrast, T_H2 cells is IL-4, which makes B cells produce IgG4 and IgE. In many experimental systems, T_H1 and T_HThe development of 2 responses is mutually exclusive, because T_H1 cell inhibition of T_H2 induction of cells and vice versa. Thus, a strong T is triggered_H1 antigen in response to simultaneous inhibition of T_H2, thereby inhibiting the production of IgE antibodies. Interestingly, virtually all viruses induce T in the host_H1 response, but not the production of IgE antibodies (Coutelier et al, J.exp. Med.165: 64-69 (1987)). This isotype profile is not limited to live viruses, but can also be observed on inactivated or recombinant viral particles (Lo-Man et al, Eur.J.Immunol.28: 1401-1407 (1998)). Thus, using the methods of the invention (e.g., alpha vaccine technology), viral particles can be loaded with a variety of allergens and used for immunization. T will be triggered due to the "viral structure" of the allergen obtained _H1 response, a "protective" IgG1-3 antibody is produced, while production of allergic IgE antibodies is prevented. Since the allergen is presented by viral particles recognized by a diverse group of helper T cells rather than the allergen itself, even when carrying an existing allergen-specific T_HAllergen-specific IgG1-3 antibodies may also be induced in 2-cell allergic individuals. The presence of high concentrations of IgG antibodies can prevent binding of allergens to mast cells that bind IgE, thereby inhibiting the release of histamine. Thus, the presence of IgG antibodies can protect against IgE-mediated allergy. Typical substances that cause allergy include: grass, ragweed, birch or cedar pollen, house dust, mites, animal dander, mold, insect venom or medicineSubstances (e.g. penicillin). It is therefore advantageous to immunize an individual with virus particles containing an allergen not only before the onset of an allergy but also after it has occurred.

In particular embodiments, the invention provides methods of preventing and/or ameliorating diseases or conditions caused or exacerbated by "self" gene products (e.g., tumor necrosis factor) (i.e., "autoantigens" as used herein). In related embodiments, the invention provides methods of inducing an immune response in an individual that results in the production of antibodies that can prevent and/or ameliorate a disease or condition caused or exacerbated by a "self" gene product. Examples of such diseases or conditions include: graft versus host disease, IgE-mediated allergy, anaphylaxis, adult respiratory distress syndrome, Crohn's disease, allergic asthma, Acute Lymphocytic Leukemia (ALL), non-Hodgkin's lymphoma (NHL), Graves 'disease, inflammatory autoimmune disease, myasthenia gravis, Systemic Lupus Erythematosus (SLE), immunoproliferative disease lymphadenopathy (IPL), angioimmunoproliferative lymphadenopathy (AIL), immunoblastic lymphadenopathy (IBL), rheumatoid arthritis, diabetes, multiple sclerosis, osteoporosis, and Alzheimer's disease.

One skilled in the art will appreciate that when a composition of the invention is administered to an individual, the composition may contain salts, buffers, adjuvants or other substances that enhance the efficacy of the composition. Examples of substances suitable for use in the preparation of PHARMACEUTICAL compositions are presented in a number of sources, including REMINGTON' S PHARMACEUTICAL SCIENCES (Osol, A., Mack Publishing Co., 1990).

A composition of the invention may be said to be "pharmacologically acceptable" if the subject is able to tolerate administration of the composition of the invention. In addition, the compositions of the present invention will be administered in a "therapeutically effective amount" (i.e., an amount that produces the desired physiological effect).

The compositions of the present invention may be administered by a variety of methods well known in the art, but are generally administered by injection, infusion, inhalation, orally, or other suitable physical means. In addition, the compositions may also be administered intramuscularly, intravenously or subcutaneously. Compositions for administration include sterile aqueous (e.g., physiological saline) or nonaqueous solutions and suspensions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. A carrier or occlusive coating/dressing may be used to increase skin permeability and enhance antigen absorption.

Prion-mediated diseases are increasingly threatening the society. In particular, prion-induced bovine BSE is a disease that has been overlooked for a long time but can affect a large number of animals throughout europe. Moreover, a variation of CJD is due to post-beef infection by humans eating prion infection. Although the number of infected persons is relatively low to date, the disease appears to be potentially epidemic. However, the long-term prognosis of vCJD development can be particularly difficult because the latency between infection and overt morbidity is extremely long (estimated at 10 years).

Prions are cellular proteins found in most mammalian species. Prion proteins exist in two forms-a normally folded form (PrP), which is normally found in healthy humans^c) And a misfolded form (Prp) causing disease^Sc). The current prion hypothesis states that the misfolded prion form Prp^ScCan catalyze healthy prion Prp^cRefolding into a pathogenic Prp^Sc(A. Aguzzi, Haematologica85, 3-10 (2000)). In some rare cases, this transformation may also occur spontaneously, causing classic CJD in humans. Prp^ScSome of the mutations in (a) are associated with this increase in spontaneous turnover, leading to different forms of familial CJD. However, Prp ^ScIt may also be infectious, and may be transmitted by blood transfusion or through the food chain. The latter type of prion-mediated disease is known as kuru and often occurs in human feeders. However, since the species that eat their own individuals are rare, the type of disease transmitted orally is very rare and is silent about other species.

Breeding mother with beef product in large amount in EuropeCattle altered BSEPrp caused by infection transmission^ScHas increased significantly in recent years, affecting hundreds of thousands of cows. The sudden appearance of a large number of BSE-bearing cows causes great panic in the population, fearing that a similar disease may be induced in humans. Indeed, in 1996 the first variant CJD was reported, probably due to food infection Prp^ScCaused by beef. This fear is still growing further to date, since the number of infections is increasing in the following years and no cure is seen. Moreover, since sheep may have a prion-mediated disease known as scrapie, other mammalian species can also infect Prp^Sc。

Experimentally, BSE-like diseases may also occur in other species. The transmission mechanism of prions has been studied in great detail. It is now clear that prions first replicate in lymphoid organs of infected mice and then transfer into the central nervous system. Follicular Dendritic Cells (FDC), a very small population of cells in lymphoid organs, appear to be essential for replication and transport of prion proteins in lymphoid organs and into the central nervous system (S.Brandner, M.A. Klein, A.Aguzzi, Transfus Clin Biol6, 17-23 (1999); F.Montrasio et al, Science288, 1257-9 (2000)). FDC is a less studied cell type, but it is now clear that their development is dependent on B cells producing lymphotoxin and/or TNF (f. mackay, j.l. browning, Nature395, 26-27 (1998)). Indeed, mice lacking lymphotoxin do not show FDC (m.s. matsumoto et al, Science264, 703-707 (1996)). Moreover, they do not infect prions effectively and do not become diseased. In addition to FDC, antibodies may also play a role in disease progression (s.brandner, m.a.klein, a.aguzzi, Transfus Clin Biol6, 17-23 (1999)).

Recently, it has been shown that blocking the Ltb pathway using the Ltb receptor Fc fusion molecule not only clears FDC in mice, but also blocks Prp^ScMontrasio et al, Science288, 1257-9 (2000)). Thus, vaccines that induce antibodies specific for LTb or its receptor may block Prp^ScFrom one toOne individual is transmitted to another or from the periphery to the central nervous system.

However, it is often difficult (or even impossible) to elicit an antibody response to the self-molecule by routine vaccination. One way to increase the efficiency of vaccination is to increase the degree of duplication of the antigens used: unlike isolated proteins, viruses can elicit a rapid, potent immune response without any adjuvant, with and without T cell help (Bachmann & Zinhkermagel, Ann. Rev. Immunol.15: 235-270 (1991)). Although viruses often contain little protein, they are able to trigger a stronger immune response than their isolated components. For B cell responses, one key factor known to be viral immutagenicity is the repetitiveness and order of surface epitopes. Many viruses display a quasi-crystalline surface displaying regularly arranged epitopes that effectively cross-link epitope-specific immunoglobulins on B cells (Bachmann & Zinkermagel, Immunol. Today17: 553-558 (1996)). This cross-linking of surface immunoglobulins on B cells is a strong activation signal that directly induces cell cycle progression and the production of IgM antibodies. Furthermore, these triggered B cells are able to activate T helper cells, which subsequently induce a switch from IgM production to IgG production, and the generation of long-term B cell memory-which is the target of all vaccination (Bachmann & Zinkemagel, Ann. Rev. Immunol.15: 235-270 (1997)). Viral structures are even involved in the production of anti-antibodies in autoimmune diseases and as part of the natural response to pathogens (see Fehr, t. et al, J exp. med.185: 1785-1792 (1997)). Thus, antibodies presented on the surface of highly organized viruses are capable of eliciting strong anti-antibody responses.

The immune system is generally unable to produce antibodies against self-structures. For soluble antigens present at low concentrations, this is due to tolerance at the Th cell level. Under these conditions, coupling of the autoantigen to a carrier capable of transporting T helper cells can break tolerance. B cells and Th cells may be tolerant to soluble proteins present at high concentrations or to membrane proteins at low concentrations. However, B cell tolerance may be reversible (anergic) and can be disrupted by administration of antigens that are coupled in a highly organized manner to foreign carriers (Bachmann & Zinkermagel, Ann. Rev. Immunol.15: 235-270 (1997)). Thus, LTb, LTa or LTb receptors, which are highly organized like viruses, virus-like particles or bacterial pili, may disrupt B cell tolerance and induce the production of antibodies specific for these molecules.

The present invention relates to the fields of molecular biology, virology, immunology and medicine. The present invention provides a method for facilitating the induction of production of specific antibodies to endogenous Lymphotoxin (LT) b, LTa or LTb receptors. The invention also provides a method for producing an antigen or epitope capable of inducing the production of antibodies specific to LTb, LTa or LTb receptors, useful for the prevention and treatment of prion-mediated diseases, such as Creutzfeldt-Jakob disease (vCJD) or Bovine Spongiform Encephalopathy (BSE), and for the clearance of lymphoid organoid structures from autoimmune diseased tissue.

The object of the present invention is to provide a vaccine capable of inducing antibodies specific to LTb, LTa or LTb receptors, thereby clearing FDC from lymphoid organs. Such treatment may prevent infection with Prp^ScOr Prp^ScSpread from the peripheral nervous system to the central nervous system. In addition, such treatment can also prevent the production of lymphoid structures in organs affected by autoimmune disease, and can solubilize existing such structures to ameliorate disease symptoms.

The LTb, LTa or LTb receptor or fragment thereof is coupled to a protein carrier that is foreign to the host. In a preferred embodiment of the invention, these molecules are coupled to highly organized structures in order to render the LTb, LTa or LTb receptor or fragment thereof highly repetitive and organized. The highly organized structure may be bacterial pili, Virus Like Particles (VLPs) produced by the following histones: recombinant protein of bacteriophage Q beta, recombinant protein of rotavirus, recombinant protein of Norwalk virus, recombinant protein of alphavirus, recombinant protein of foot-and-mouth disease virus, recombinant protein of retrovirus, recombinant protein of hepatitis B virus, recombinant protein of tobacco mosaic virus, recombinant protein of poultry hut virus and recombinant protein of human papilloma virus. To optimize the three-dimensional arrangement of the LTb, LTa or LTb receptor or fragment thereof on a highly organized structure, attachment sites, such as chemically reactive amino acids, are introduced into the highly organized structure (unless naturally occurring), and binding sites, such as chemically reactive amino acids, are introduced on the LTb, LTa or LTb receptor or fragment thereof (unless naturally occurring). The presence of attachment sites on highly organized structures and binding sites on LTb, LTa or LTb receptors or fragments thereof will allow these molecules to couple to repetitive structures in a directed, ordered manner, which is necessary to elicit an effective B cell response.

In an equally preferred embodiment, the attachment site introduced into the repetitive structure is biotin which specifically binds streptavidin. Biotin can be introduced by chemical modification. The LTb, LTa or LTb receptor or fragment thereof can be fused or linked to streptavidin and bound to biotinylated repeats.

Other embodiments of the invention include methods of producing the compositions of the invention and methods of medical treatment using these compositions. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.

In addition to vaccine technology, other embodiments of the present invention relate to methods for the medical treatment of cancer and allergies.

All patents and publications mentioned herein are incorporated by reference in their entirety.

Examples

Enzymes and reagents used in the following experiments included: t4 DNA ligase obtained from NewEngland Biolabs; taq DNA polymerase, QIAprep Spin plasmid kit, QIAGEN plasmid Midi kit, QiaExII gel extraction kit, QIAquick PCR purification kit, all obtained from QIAGEN; QuickPrep Micro mRNA purification kit, obtained from Pharmacia; SuperScript one-step RT PCR kit, Fetal Calf Serum (FCS), bacto tryptone, and yeast extract, obtained from Gibco BRL; oligonucleotides, available from Microsynth (switzerland); restriction endonucleases, available from Boehringer Mannheim, NewEngland Biolabs or MBI Fermentas; pwo polymerase and dNTPs from Boehringer Mannheim. HP-1 medium was obtained from Cell Culture Technologies (Glattbrugg, Switzerland). All standard chemicals were obtained from Fluka-Sigma-Aldrich and all cell culture material from TPP.

DNA manipulations were performed according to standard techniques. DNA was prepared from 2ml of bacterial culture using the QIAprep Spin plasmid kit or 50ml of culture using the QIAGEN plasmid Midi kit according to the instructions. For restriction enzyme digestion, the DNA is incubated with an appropriate restriction enzyme at a concentration of 5-10 units (U) enzyme/mgDNA for at least 2 hours under the manufacturer's recommended conditions (buffer and temperature). If the reaction conditions are suitable for all enzymes, the digestion is carried out simultaneously with more than one enzyme or successively. For further manipulation, the digested DNA fragments were separated by 0.7-1.5% agarose gel electrophoresis, excised from the gel, and purified using the QiaExII gel extraction kit according to the manufacturer's instructions. For ligation of DNA fragments, 100-200pg of purified vector DNA was incubated overnight with 3-fold molar excess of insert in the buffer provided by the manufacturer (total volume: 10-20. mu.l) at 16 ℃ in the presence of 1U T4 DNA ligase. Coli XL1-Blue (Stratagene) was transformed with one aliquot (0.1-0.5. mu.l) of ligation. Transformation was performed by electroporation using Gene Pulser (BioRAD) and a 0.1cm Gene Pulser cell (BioRAD) at 200Ohm, 25. mu.F, 1.7 kV. After electroporation, cells were incubated for 1 hour in 1ml of s.o.b. medium (Miller, 1972) with shaking, after which they were plated on selective s.o.b. agar.

Example 1

Modular eukaryotic expression system for antigen-to-VLP coupling

This system is created by adding different amino acid linker sequences containing cysteine residues to the antigen for chemical coupling to the VLPs.

A. Construction of an EBNA-derived expression System encoding a linker containing cysteine amino acids and a cleavable Fc-Tail

pCep-Pu (Wuttke et al, J.biol.chem.276: 36839-48(2001)) was digested with Kpn I and Bam HI, and a new multiple cloning site was introduced using annealed oligonucleotides PH37(SEQ ID NO: 270) and PH38(SEQ ID NO: 271), to generate pCep-MCS.

A modular system containing one free cysteine flanked by several glycines, one protease cleavage site, and a human IgG1 constant region was generated as follows. pSec2/Hygro B (Invitrogen Cat. No. V910-20) was digested with Bsp120I and HindIII and ligated with the annealed oligonucleotides SU7(SEQ ID NO: 278) and SU8(SEQ ID NO: 279) to generate construct pSec-B-MCS. pSec-B-MCS was then digested with NheI and HindIII and ligated with the annealed oligonucleotides PH29(SEQ ID NO: 264) and PH30(SEQ ID NO: 265) to generate construct pSec 29/30. Construct pSec-FL-EK-Fc^*Generated by ligation of the following three fragments: first pSee29/30 digested with Eco RI and Hind III, annealed oligonucleotides PH31(SEQ ID NO: 266) and PH32(SEQ ID NO: 267), and a plasmid containing a modified form of the human IgG1 constant region (pSP-Fc) ^*-C1) (details of the hu IgG1 sequence see final construct pCep-Xa-Fc)^*1A-1C). pCep-Xa-Fc^*The complete sequence of (a) is as set forth in SEQ ID NO: 283 are listed. The resulting construct was named pSec-FL-EK-Fc^*. This plasmid was digested by Nhe I/Pme I, the linker region and the human IgG1 Fc portion were excised from the plasmid, cloned into the pCep-MCS digested with Nhe I and Pme I, and the construct pCep-FL-EK-Fc was generated^*. This results in a modular vector in which the linker sequence and protease cleavage site are located between the Nhe I and Hind III sites, which are readily exchanged for the annealed oligonucleotide. To generate a cleavable fusion protein, vector pCep-FL-EK-F was digested with Nhe I and Hind III^*The factor Xa cleavage site with the amino acid GGGGCG on the N-terminal side was introduced with the annealed oligonucleotides PH35(SEQ ID NO: 268) and PH36(SEQ ID NO: 269), and the annealed oligonucleotides PH39(SEQ ID NO: 272) and PH36PH40(SEQ ID NO: 273) was introduced into an enterokinase site flanked at the N-terminus by GGGGCG to generate constructs pCep-Xa-Fc^*(see FIG. 1A) and pCep-EK-Fc^*(see FIG. 1B). In addition, the construct pCep-SP-EK-Fc containing eukaryotic signal peptide^*(see FIG. 1C) was generated by ligation of the following three fragments: kpn I/Bam HI digested pCep-EK-Fc ^*Annealed oligonucleotides PH41(SEQ ID NO: 274) and PH42(SEQ ID NO: 275), and annealed oligonucleotides PH43(SEQ ID NO: 276) and PH44(SEQ ID NO: 277).

B. Large-Scale production of fusion proteins

For large-scale preparation of different fusion proteins, 293-EBNA cells (Invitrogen) were transfected with different pCep expression plasmids using Lipofectamine2000 reagent (Life technologies) according to the manufacturer's recommendations. 24-36 hours after transfection, cells were plated at a 1: 3 ratio in DMEM supplemented with 10% FCS under puromycin (1. mu.g/ml) selection. The drug-resistant cells are then expanded in selection medium. To harvest the fusion protein, the drug-resistant cell population was passaged onto poly-L-lysine-coated dishes. Once the cells were confluent, they were washed 2 times with PBS and serum free medium (DMEM) was added to the plates. Tissue culture supernatants were collected every 2-4 days for a period up to 1 month and replaced with fresh DMEM medium. The harvested supernatant was stored at 4 ℃.

C. Purification of fusion proteins

The recombinant Fc-fusion protein was purified by affinity chromatography using protein A Sepharose CL-4B (Amersham pharmacia Biotech AG). Briefly, 1-3ml of protein A resin was packed onto a chromatography column, and a tissue culture supernatant containing the recombinant protein was applied to the column at a flow rate of 0.5-1.5ml/min using a peristaltic pump. The column was then washed with 20-50ml PBS. Depending on the fusion protein, protease cleavage is performed on the column, or the protein is eluted as described below. The recombinant fusion protein was eluted with a citric acid/phosphate buffer (pH3.8) containing 150mM NaCl, and the protein-containing fractions were combined and concentrated with an ultrafree centrifugal filter (Millipore).

D. Protease cleavage of recombinant fusion proteins (factor Xa, enterokinase)

The eluted recombinant fusion protein containing the Enterokinase (EK) cleavage site was cleaved using the EKmax system (Invitrogen) according to the manufacturer's recommendations. The Fc portion of the cleaved fusion protein was removed by incubation with protein a. The enterokinase was then removed using the EK-Away system (Invitrogen) according to the manufacturer's recommendations. Similarly, fusion proteins containing a factor Xa (Xa) cleavage site were cleaved by the restriction protease factor Xa cleavage and removal kit (Roche) according to the manufacturer's recommendations. The cleaved Fc portion was removed by incubation with protein A, and the protease was removed with the kit-attached streptavidin resin.

The different fusion proteins were concentrated using ultrafree centrifugal filters (Millipore) and quantified by uv spectrophotometry for subsequent coupling reactions.

FIGS. 1A-1C show partial sequences of different eukaryotic expression vectors used. Only the modified sequence is shown.

FIG. 1A: pCep-Xa-Fc^*: the sequences are shown forward from the BamHI site, with different features shown above the translated sequence. The arrow indicates the factor Xa protease cleavage site.

FIG. 1B: pCep-EK-Fc^*: the sequences are shown forward from the BamHI site, with different features shown above the translated sequence. The arrow indicates the enterokinase cleavage site. The sequence downstream of the Hind III site is the same as shown in FIG. 1A.

FIG. 1C: pCep-SP-EK-Fc^*: the sequence is shown from the beginning of the signal peptide onwards, with different features shown above the translated sequence. The signal peptide sequence cleaved with the signal peptide is shown in bold. The arrow indicates the enterokinase cleavage site. The sequence downstream of the Hind III site is the same as shown in FIG. 1A.

Example 2

Eukaryotic expression of mouse resistin and coupling to VLPs and pili

A. Cloning of mouse resistin

Total RNA was isolated from 60mg mouse adipose tissue using Qiagen RNeasy kit according to the manufacturer's recommendations. Using 40 mu l H₂And O, eluting the RNA. Reverse transcription was then performed with the total RNA, using oligo dT primer and ThermoScopt RT-PCR system (Life technologies), according to the manufacturer's recommendations. The samples were incubated at 50 ℃ for 1 hour, heated to 85 ℃ for 5 minutes and treated with RNAseH at 37 ℃ for 20 minutes.

PCR amplification of mouse resistin was performed using 2. mu. lRT reaction. PCR was performed with platinum TAQ (Life Technologies) according to the manufacturer's recommendations using primers PH19(SEQ ID NO: 260) and PH20(SEQ ID NO: 261). Primer PH19(SEQ ID NO: 260) corresponds to positions 58-77 of the mouse resistin sequence and primer PH20(SEQ ID NO: 261) corresponds to positions 454-435. The PCR mixture was first denatured at 94 ℃ for 2 minutes, then subjected to 35 cycles as follows: 94 ℃ for 30 seconds, 56 ℃ for 30 seconds, 72 ℃ for 1 minute, and finally the sample was left at 72 ℃ for 10 minutes. The PCR fragment was purified and subcloned into pGEMTeasy vector (Invitrogen) by TA cloning to generate pGEMT-mRes. To add the appropriate restriction sites, pGEMT-mRes was subjected to a second PCR with primers PH21(SEQ ID NO: 262) and PH22(SEQ ID NO: 263) using the same cycling protocol as described above. The forward primer (PH21(SEQ ID NO: 262)) contained a Bam HI site and nucleotides 81-102 of the mouse resistin sequence. The reverse primer (PH22(SEQ ID NO: 263)) contained an Xba I site and nucleotides 426-406 of the mouse resistin sequence. The locus is referenced to the mouse resistin sequence gene accession number AF 323080. The PCR product was purified, digested with Bam HI and Xba I, subcloned into BamHI and Xba I digested pcmv-Fc ^*-C1, producing the construct pcmv-mRes-Fc^*。

From pcmv-mRes-Fc by Bam HI/Xba I digestion^*The open reading frame for resistin was excised and cloned into Bam HI and Nhe I digested pCep-Xa-Fc^*And pCep-EK-Fc^*(see example 1, part B), respectively, to generate the construct pCep-mRes-Xa-Fc^*And pCep-mRes-EK-Fc^*。

B. Preparation, purification and cleavage of resistin

Then using pCep-mRes-Xa-Fc^*And pCep-mRes-EK-Fc^*The constructs were transfected into 293-EBNA cells to produce recombinant proteins as described in example 1, part B. Tissue culture supernatants were purified as described in example 1 part C. The purified protein was then cleaved as described in example 1, part D. The recombinant proteins produced were designated "resistin-C-Xa" or "Res-C-Xa" and "resistin-C-EK" or "Res-C-EK", depending on the vector used (see FIG. 2A and FIG. 2B).

Fig. 2A and 2B show the sequences of recombinant mouse resistin proteins for expression and further conjugation. Res-C-Xa (FIG. 2A) and Res-C-EK (FIG. 2B) show the translated DNA sequences. The resistin signal sequence cleaved by the signal peptidase following secretion of the protein is shown in italics. The amino acid sequences resulting from cleavage by signal peptidases and specific proteases (factor Xa or enterokinase) are shown in bold. The bold sequence corresponds to the amino acid protein sequence actually used for coupling, i.e. SEQ id no: 280 and SEQ ID NO: 281. SEQ ID NO: 282 corresponds to another resistin protein construct which can also be used to couple to virus like particles and pili according to the invention.

C. Coupling of resistin-C-Xa and resistin-C-EK to Q beta capsid proteins

0.2ml of a solution of 2mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 5.6. mu.l of a solution of 100mM SMPH (Pierce) in DMSO on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed Q.beta.reaction mixture was reacted with 32. mu.l of a resistin-C-Xa solution (giving a final concentration of 0.39mg/ml resistin), 13. mu. l Q. beta.reaction mixture and 27. mu.l of a resistin-C-EK solution (giving a final concentration of 0.67mg/ml resistin) on a shaker at 25 ℃ for 4 hours. The coupled product was analyzed by SDS-PAGE (see FIG. 2C). There was another band of 24kDa in the coupling reaction but not in the derivatized Q.beta.and resistin. The 24kDa size corresponds to the expected size of the conjugate product 24kDa (14kDa Q.beta.plus 10kDa resistin-C-Xa and resistin-C-EK, respectively).

FIG. 2C shows the results of coupling of resistin-C-Xa and resistin-C-EK to Q β. The coupled products were analyzed on a 16% SDS-PAGE gel under reducing conditions. Lane 1: and (4) molecular weight standard. And (2) a step: resistin-C-EK prior to conjugation. And (3) a step: the coupled resistin-C-EK-Q beta. And 4, a 4 th step: derivatized Q β. And (5) a step: resistin-C-Xa before coupling. And 6, a step of: coupled resistin-C-Xa-Q beta. Molecular weights of the standard proteins are shown in the left blank. The coupling bands are indicated by arrows.

D. Conjugation of resistin-C-Xa and resistin-C-EK to fr capsid proteins

2mg/ml fr capsid protein in 0.2ml 20mM Hepes, 150mM NaCl pH7.4 was reacted with 5.6. mu.l of 100mM SMPH (Pierce) in DMSO on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed fr capsid protein reaction mixture was reacted with 32. mu.l of a resistin-C-Xa solution (giving a final concentration of 0.39 mg/ml), 13. mu. lfr of the capsid protein reaction mixture and 27. mu.l of a resistin-C-EK solution (giving a final concentration of 0.67 mg/ml) for 4 hours at 25 ℃ on a shaker. The coupled products were analyzed by SDS-PAGE under reducing conditions.

E. Conjugation of resistin-C-Xa and resistin-C-EK to HBcAg-Lys-2cys-Mut

0.2ml of a solution of 2mg/ml HBcAg-Lys-2cys-Mut in 20mM Hepes, 150mM NaCl pH7.2 was reacted with 5.6. mu.l of a solution of 100mM SMPH (Pierce) in DMSO on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed HBcAg-Lys-2cys-Mut reaction mixture was reacted with 32. mu.l of resistin-C-Xa solution, 13. mu.l of HBcAg-Lys-2cys-Mut reaction mixture and 27. mu.l of resistin-C-EK solution on a shaker at 25 ℃ for 4 hours. The coupled products were analyzed by SDS-PAGE.

F. Coupling of resistin-C-Xa and resistin-C-EK to pili

Mu.l of a solution of 2.5mg/ml E.coli type 1 fimbriae in 20mM Hepes pH7.4 was reacted with a 50-fold molar excess of the cross-linking agent SMPH (Pierce) diluted from DMSO stock solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted by passing through a PD-10 column (Amersham-Pharmacia Biotech). The protein-containing fractions eluted from the column were combined, and 8. mu.l of the desalted derivative pilin was reacted with 32. mu.l of the resistin-C-Xa solution, 13. mu.l of the desalted derivative pilin was reacted with 27. mu.l of the resistin-C-EK solution on a shaker at 25 ℃ for 4 hours. The coupled products were analyzed by SDS-PAGE.

Example 3

A. Introduction of cysteine-containing linker, expression and purification of mouse lymphotoxin-beta

Recombinantly expressing the extracellular portion of mouse lymphotoxin- (LT-), which contains a CGG amino acid linker at the N-terminus. The linker contains a cysteine for coupling to the VLP. For ease of purification, the long (amino acids 49-306) and short (amino acids 126-306) forms of the protein are fused at the N-terminus to glutathione S-transferase (GST) or histidine-myc tails. To cut the tail, an Enterokinase (EK) cleavage site was inserted.

Construction of C-LT 49-306 and C-LT 126-306

Mouse LT.49-306 was amplified from a mouse spleen cDNA library inserted into pFB-LIB using oligonucleotides 5 'LT. and 3' LT. as primers by PCR. For the PCR reaction, 50. mu.l of the reaction mixture (1 unit PFX Platinum polymerase, 0.3mM dNTPs and 2mM MgSO)₄) 0.5. mu.g each of the primers and 200ng of the template DNA were used. The temperature cycle was as follows: 94 ℃ for 2 min, then 94 ℃ (15 sec), 68 ℃ (30 sec), 68 ℃ (1 min) 25 cycles, followed by 68 ℃ for 10 min. The PCR product was phosphorylated with T4 kinase and ligated into EcoRV digested and dephosphorylated pEntryA (Life Technologies). The resulting plasmid was designated as pEntryA-LT 49-306.

A second PCR reaction was carried out using pEntryl A-LT 49-306 as a template and oligonucleotides 5 'LT long-NheI and 3' LT stop-NotI and 5 'LT short-NheI and 3' LT stop-NotI, respectively. The oligonucleotides 5 'LT long-NheI and 5' LT short-NheI contain an internal NheI site,and contains a codon for a Cys-Gly-Gly linker, and 3' LT.stop-NotI contains an internal NotI site and contains a stop codon. For the second PCR reaction, 50. mu.l of reaction mixture (1 unit PFX Platinum polymerase, 0.3mM dNTPs and 2mM MgSO) ₄) 0.5. mu.g each of the primers and 150ng of the template DNA were used. The temperature cycle was as follows: 94 ℃ for 2 minutes, then 94 ℃ (15 seconds), 50 ℃ (30 seconds), 68 ℃ (1 minute) for 5 cycles, then 94 ℃ (15 seconds), 64 ℃ (30 seconds), 68 ℃ (1 minute) for 20 cycles, followed by 68 ℃ for 10 minutes.

The PCR product was digested with NheI and NotI and inserted into pCEP-SP-GST-EK or pCEP-SP-his-myc-EK (Wuttke et al, J.biol.chem.276: 36839-48 (2001)). The resulting plasmids were designated pCEP-SP-GST-EK-C-LT 49-306, pCEP-SP-GST-EK-C-LT 126-306, pCEP-SP-his-myc-EK-C-LT 49-306, and pCEP-SP-hiS-myc-EK-C-LT 126-306, respectively. GST is glutathione-S-transferase, EK is enterokinase, his is hexa-histidine tail, myc is anti-c-myc epitope. C represents a CGG linker further comprising a cysteine.

All other steps were performed according to standard molecular biology methods.

The oligonucleotide sequence:

5’LT·：

5’-CTT GGT GCC GCA GGA TCA G-3’(SEQ ID NO：284)

3’LT·：

5’-CAG ATG GCT GTC ACC CCA C-3’(SEQ ID NO：285)

5’LT·long-NheI：

5’-GCC CGCTAG CCT GCG GTG GTC AGG ATC AGG GAC GTC G-3’(SEQID NO：286)

5’LT·short-NheI：

5’-GCC CGC TAG CCT GCG GTG GTT CTC CAG CTG CGG ATT C-3’(SEQID NO：287)

3’LT·stop-NotI：

5’-CAA TGA CTG CGG CCG CTT ACC CCA CCA TCA CCG-3’(SEQ ID NO：288)。

GST-EK-C-LT·_49-306、GST-EK-C-LT·_126-306、his-myc-EK-C-LT·_49-306and his-myc-EK-C-LT_126-306Expression and preparation of

To prepare the protein as described in example 1, plasmids were used

pCEP-SP-GST-EK-C-LT·49-306、pCEP-SP-GST-EK-C-LT·126-306、

pCEP-SP-his-myc-EK-C-LT 49-306 and

pCEP-SP-his-myc-EK-C-LT 126-306 transfected 293-EBNA cells (Invitrogen).

The prepared protein was named GST-EK-C-LT_49-306、GST-EK-C-LT·_126-306、his-myc-EK-C-LT·_49-306And his-myc-EK-C-LT_126-306。

The protein sequence of the LT-fusion protein is translated from the cDNA sequence:

GST-EK-C-LT·_49-306：SEQ ID NO：289

GST-EK-C-LT·_126-306：SEQ ID NO：290

his-myc-EK-C-LT·_49-306：SEQ ID NO：291

his-myc-EK-C-LT·_126-306：SEQ ID NO：292

The fusion proteins were analyzed on 12% SDS-PAGE under reducing conditions. The gel was blotted onto nitrocellulose membrane. The membrane was blocked and incubated with either monoclonal mouse anti-myc antibody or with anti-GST antibody. The blot was then incubated with either horseradish peroxidase-conjugated goat anti-mouse IgG or horseradish peroxidase-conjugated rabbit anti-goat IgG. The results are shown in fig. 3. GST-EK-C-LT_49-306And GST-EK-C-LT_126-306Detectable molecules with anti-GST antibodiesThe amounts were 62kDa and 48kDa, respectively. his-myc-EK-C-LT_49-306And his-myc-EK-C-LT_126-306Can be detected with anti-myc antibodies, 40-56kDa and 33-39kDa, respectively.

FIGS. 3A and 3B show the results of the expression of LT & fusion proteins, which were analyzed on 12% SDS-PAGE under reducing conditions. The gel was blotted onto nitrocellulose membrane. The membrane was blocked and incubated with either monoclonal mouse anti-myc antibody (1: 2000 dilution) (FIG. 3A) or anti-GST antibody (1: 2000 dilution) (FIG. 3B). The blot was then incubated with either horseradish peroxidase-conjugated goat anti-mouse IgG (1: 4000 dilution) (FIG. 3A) or horseradish peroxidase-conjugated rabbit anti-goat IgG (1: 4000 dilution) (FIG. 3B). A: lanes 1 and 2: his-myc-EK-C-LT_126-306. Lanes 3 and 4: his-myc-EK-C-LT _49-306. B: lanes 1 and 2: GST-EK-C-LT_126-306. Lanes 3 and 4: GST-EK-C-LT_49-306. Molecular weights of the standard proteins are shown in the left blank.

B.GST-EK-C-LT·_49-306、GST-EK-C-LT·_126-306、his-myc-EK-C-LT·_49-306And his-myc-EK-C-LT_126-306Purification of (2)

GST-EK-C-LT·_49-306And GST-EK-C-LT_126-306Purification on a glutathione-Sepharose column, his-myc-EK-C-LT_49-306And his-myc-EK-C-LT_126-306Purification was performed using a Ni-NTAsepharose column, all using standard purification methods. The purified protein was cleaved with enterokinase and analyzed on a 16% SDS-PAGE gel under reducing conditions.

C.C-LT·_49-306And C-LT ·_126-306Conjugation to Q beta capsid protein

A solution of 120. mu.MQ β -capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock solution on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. Dialyzed Q beta reaction mixture with C-LT_49-306And C-LT ·_126-306Solution (final concentration:60μM Qβ，60μM C-LT·_49-306and C-LT ·_126-306) The reaction was carried out on a shaker at 25 ℃ for 4 hours. The coupled products were analyzed by SDS-PAGE.

D.C-LT·_49-306And C-LT ·_126-306Conjugation to fr capsid proteins

A solution of 120. mu.M fr capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock solution for 30 min at 25 ℃ on a shaker. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. Dialyzed fr capsid protein reaction mixture with C-LT _49-306And C-LT ·_126-306Solution (final concentration: 60. mu.M fr, 60. mu. M C-LT. cndot.)_49-306And C-LT ·_126-306) The reaction was carried out on a shaker at 25 ℃ for 4 hours. The coupled products were analyzed by SDS-PAGE under reducing conditions.

E.C-LT·_49-306And C-LT ·_126-306Coupling with HBcAg-Lys-2cys-Mut

A solution of 120. mu.M HBcAg-Lys-2cys-Mut capsid in 20mM Hepes, 150mM NaClpH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock for 30 min at 25 ℃ in a shaker. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. Dialyzed HBcAg-Lys-2cys-Mut reaction mixture with C-LT_49-306And C-LT ·_126-306Solution (final concentration: 60. mu. MHBcAG-Lys-2cys-Mut, 60. mu. M C-LT. cndot. (g)_49-306And C-LT ·_126-306) The reaction was carried out on a shaker at 25 ℃ for 4 hours. The coupled products were analyzed by SDS-PAGE.

F.C-LT·_49-306And C-LT ·_126-306Coupling to pili

A solution of 125. mu.M E.coli type 1 pili in 20mM Hepes pH7.4 was reacted with a 50-fold molar excess of the cross-linking agent SMPH (Pierce) diluted in DMSO stock solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted using a PD-10 column (Amersham-Pharmacia Biotech). Combining the protein-containing fractions eluted from the column, desalting the pooled fractions to obtain a derivative pilinWhite with C-LT_49-306And C-LT ·_126-306Solution (final concentration: 60. mu.M pilus, 60. mu. M C-LT. cndot.) _49-306And C-LT ·_126-306) The reaction was carried out on a shaker at 25 ℃ for 4 hours. The coupled products were analyzed by SDS-PAGE under reducing conditions.

Example 4

A. Introduction of cysteine-containing linker, expression, purification and coupling of rat macrophage migration inhibitory factor MIF with Q beta

The rat macrophage migration inhibitory factor (rMIF) was recombinantly expressed fusing three different amino acid linkers C1, C2, and C3 at the C-terminus. Each linker contains a cysteine for coupling to the VLP.

Construction of rMIF-C1, rMIF-C2 and rMIF-C3

The MCS for pET22b (+) (Novagen, Inc.) was changed to GTTTAACTTTAAGAAGGAGATATACATATGGATCCGGCTAGCGCTCGAGGGTTTAAACGGCGGCCGCATGCACC by replacing the original sequence between the NdeI site and the XhoI site with annealed oligonucleotide primers MCS-1F and MCS-1R (annealed in 15mM TrisHCl pH8 buffer). The resulting plasmid, designated pMod00, contained NdeI, BamHI, NheI, XhoI, PmeI and NotI restriction sites in the MCS. The annealed BamHi 6-EK-Nhe-F and BamHi 6-EK-Nhe-R oligonucleotide pairs were ligated together with annealed oligo 1F-C-glycine-linker and oligo 1R-C-glycine-linker pairs into the BamHI-NotI digested pMod00 plasmid to generate pModeCl, which contained an N-terminal hexahistidine tail, an enterokinase cleavage site and a C-terminal amino acid glycine linker containing a cysteine residue. The annealed BamHi 6-EK-Nhe-F and BamHi 6-EK-NheR oligonucleotide pairs were ligated together with annealed oligo1F-C- γ 1-linker and oligo1R-C- γ 1-linker pairs into the BamHI-NotI digested pMod00 plasmid to generate pModeEC 2, which contained an N-terminal hexahistidine tail, an enterokinase cleavage site and a C-terminal.1 linker derived from the human immunoglobulin γ 1 hinge region and containing a cysteine residue. The annealed BamHi 6-EK-Nhe-F and BamHi 6-EK-NheR oligonucleotide pair, annealed oligo1FA-C- γ 3-linker and oligo1RA-C- γ 3-linker pair and annealed oligo1FB-C- γ 3-linker and oligo1RB-C- γ 3-linker pair were ligated together into a BamHI-NotI digested pMod00 plasmid to yield pModeEC 3, which contained an N-terminal hexahistidine tail, an enterokinase cleavage site and a C-terminal 3 linker derived from the mouse immunoglobulin-3 hinge region and containing a cysteine residue.

pBS-rMIF containing rat MIFcDNA was amplified by PCR using the oligonucleotides rMIF-F and rMIF-Xho-R. rMIF-F contains an internal NdeI site and rMIF-Xho-R contains an internal XhoI site. The PCR products were digested with NdeI and XhoI and ligated into pModEC1, pModEC2, and pModEC3 digested with the same enzymes. The resulting plasmids were designated pMod-rMIF-C1, pMod-rMIF-C2 and pMod-rMIF-C3, respectively.

For the PCR reaction, the reaction mixture (2 units PFX polymerase, 0.3mM dNTPs and 2mM MgSO 2. sup.1) was subjected to 50. sup.1 reaction₄) 15pmol each of the primers and 1ng of the template DNA were used. The temperature cycle was as follows: 94 ℃ for 2 minutes, then 94 ℃ (30 seconds), 60 ℃ (30 seconds), 68 ℃ (30 seconds) for 30 cycles, followed by 68 ℃ for 2 minutes.

All other steps were performed according to standard molecular biology methods.

The oligonucleotide sequence:

primer MCS-1F:

5’-TAT GGA TCC GGC TAG CGC TCG AGG GTT TAA ACG GCG GCC GCAT-3’(SEQ ID NO：293)

primer MCS-1R:

5’-TCG AAT GCG GCC GCC GTT TAA ACC CTC GAG CGC TAG CCG GATCCA-3’(SEQ ID NO：294)

Bamhis6-EK-Nhe-F：

5’-GAT CCA CAC CAC CAC CAC CAC CAC GGT TCT GGT GAC GAC GATGAC AAA GCG CTA GCC C-3’(SEQ ID NO：295)

Bamhis6-EK-Nhe-R：

5’-TCG AGG GCT AGC GCT TTG TCA TCG TCG TCA CCA GAA CCG TGGTGG TGG TGG TGG TGT G-3’(SEQ ID NO：296)

olio 1F-C-glycine-linker:

5’-TCG AGG GTG GTG GTG GTG GTT GCG GTT AAT AAG TTT AAACGC-3’(SEQ ID NO：297)

oligo 1R-C-glycine-linker:

5’-GGC CGC GTT TAA ACT TAT TAA CCG CAA CCA CCA CCA CCACCC-3’(SEQ ID NO：298)

oligo1F-C- γ 1-linker:

5’-TCG AGG AAA CCC ACA CCT CTC CGC CGT GTG GTT AAT AAGTTT AAA CGC-3’(SEQ ID NO：299)

oligo1R-C- γ 1-linker:

5’-GGC CGC GTT TAA ACT TAT TAA CCA CAC GGC GGA GAG GTG TGGGTT TTA TCC-3’(SEQ ID NO：300)

oligo1FA-C- γ 3-linker:

5’-TCG AGC CGA AAC CGT CTA CCC CGC CGG GTT CTT CTG-3’(SEQ IDNO：301)

oligo1RA-C- γ 3-linker:

5’-CAC CAC CAG AAG AAC CCG GCG GGG TAG ACG GTT TCG GC-3’(SEQ ID NO：302)

oligo2FB-C- γ 3-linker:

5’-GTG GTG CTC CGG GTG GTT GCG GTT AAT AAG TTT AAA CGC-3’(SEQ ID NO：303)

oligo2RB-C- γ 3-linker:

5’-GGC CGC GTT TAA ACT TAT TAA CCG CAA CCA CCC GGA G-3’(SEQID NO：304)

rMIF-F：

5’-GGA ATT CCA TAT GCC TAT GTT CAT CGT GAA CAC-3’(SEQ IDNO：305)

rMIF-Xho-R：

5’-CCC GCT CGA GAG CGA AGG TGG AAC CGT TC-3’(SEQ ID NO：306)

expression and purification of rMIF-C

Competent E.coli BL21(DE3) cells were transformed with plasmids pMod-rMIF-C1, pMod-rMIF-C2 and pMod-rMIF-C3. Individual colonies on ampicillin-containing (Amp) agar plates were expanded in liquid medium (SB with 150mM MOPS, pH7.0, 200. mu.g/ml Amp, 0.5% glucose) and incubated overnight at 30 ℃ with shaking at 220 rpm. Then 1L of SB (150mM MOPS, pH7.0, 200. mu.g/ml Amp) was inoculated with 1: 50v/v of overnight culture medium and cultured at 30 ℃ until OD600 became 2.5. Expression was induced with 2mM IPTG. Cells were harvested after overnight culture and centrifugation at 6000 rpm. The cell pellet was suspended in lysis buffer (10mM Na) containing 0.8mg/ml lysozyme ₂HPO₄30mM NaCl, 10mM EDTA and 0.25% Tween-20), sonicated and treated with benzonase. 2ml of lysate were then passed through a 20ml Q XL-column and a 20ml SP XL-column. Proteins rMIF-C1, rMIF-C2 and rMIF-C3 flow out.

The protein sequence of rMIF-C is translated from the cDNA sequence.

rMIF-C1：SEQ ID NO：307

rMIF-C2：SEQ ID NO：308

rMIF-C3：SEQ ID NO：309

Coupling of rMIF-C1 to Q capsid protein

A solution of 6mg/ml Q.capsid protein in 20mM Hepes, 150mM NaCl pH7.2 (1.48 ml) was reacted with 14.8. mu.l of SMPH (Pierce) from a 100mM stock solution dissolved in DMSO at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM hepes, 150mM NaCl pH7.0 at 4 ℃ for 3 hours each time. 3.6 mg/ml rMIF-C1 protein in 20mM Hepes, 150m1.3ml of a solution of M NaCl in pH7.2 was mixed with 9.6. mu.l of TCEP (Pierce) (from solution in H)₂O36 mM stock solution) was reacted at 25 ℃ for 1 hour. Mu.l of derivatized dialyzed Q.was reacted with 129. mu.l of reduced rMIF-C1 in 241. mu.l of 20mM Hepes, 150mM NaCl pH7.0 overnight at 25 ℃.

Coupling of rMIF-C2 to Q capsid protein

0.9ml of a solution of 5.5mg/ml Q.capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with 9. mu.l of SMPH (Pierce) from a 100mM stock solution dissolved in DMSO at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. 850. mu.l of a solution of 5.80mg/ml rMIF-C2 protein in 20mM Hepes, 150mM NaCl pH7.2 was mixed with 8.5. mu.l of TCEP (Pierce) (from H solution) ₂O36 mM stock solution) was reacted at room temperature for 1 hour. Mu.l of derivatized dialyzed QX was reacted with 85. mu.l of reduced rMIF-C2 in 335. mu.l of 20mM Hepes, 150mM NaCl pH7.2 overnight at 25 ℃.

Coupling of rMIF-C3 to Q capsid protein

A solution of 6mg/ml Q.capsid protein in 20mM Hepes, 150mM NaCl pH7.2 (1.48 ml) was reacted with 14.8. mu.l of SMPH (Pierce) from a 100mM stock solution dissolved in DMSO at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM hepes, 150mM NaCl pH7.0 at 4 ℃ for 2 times 3 hours. 720. mu.l of a solution of 5.98mg/ml rMIF-C3 protein in 20mM Hepes, 150mM NaCl pH7.2 was mixed with 9.5. mu.l of TCEP (Pierce) (from H solution)₂O36 mM stock solution) was reacted at 25 ℃ for 1 hour. Mu.l derivatized and dialyzed Q.80. mu.l reduced rMIF-C3 was reacted overnight at 25 ℃ in 290. mu.l 20mM Hepes, 150mM NaCl pH 7.0.

All three coupled products were analyzed on a 16% SDS-PAGE gel under reducing conditions. The gel was stained with Coomassie Brilliant blue or blotted onto nitrocellulose. The membranes were blocked and incubated with polyclonal rabbit anti-Qb antiserum (1: 2000 dilution) or purified rabbit anti-MIF antibody (Torrey Pines Biolabs, Inc.) (1: 2000 dilution). The blot was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1: 2000 dilution). The results are shown in fig. 4A and 4B. The conjugate product was detected in a coomassie stained gel (fig. 4A), and covalent conjugation of all three rMIF variants to Q β capsid protein was clearly demonstrated with anti-Q β anti-serum and anti-MIF antibodies (fig. 4B).

Fig. 4A shows coupling of MIF constructs to Q β. The coupled products were analyzed on a 16% SDS-PAGE gel under reducing conditions. The gel was stained with Coomassie Brilliant blue. Molecular weights of the standard proteins are shown in the left blank.

FIG. 4B shows the coupling of MIF-C1 to Q β. The coupled products were analyzed on a 16% SDS-PAGE gel under reducing conditions. Lane 1: MIF-C1 before coupling. And (2) a step: derivatized Q β prior to coupling. Lanes 3-5: q beta-MIF-C1. Lanes 1-3 were stained with Coomassie Brilliant blue. Lanes 4 and 5 are Western blots of coupled reactions developed with anti-MIF antiserum and anti-Q β antiserum, respectively. Molecular weights of the standard proteins are shown in the left blank.

B. Immunization of mice with MIF-C1 coupled to Q β capsid protein

Female Balb/C mice were vaccinated with MIF-C1 coupled to Q β capsid protein without adjuvant. 25 μ g of total protein per sample was diluted to 200 μ l with PBS and injected subcutaneously (100ml, on both sides of the abdomen) on days 0 and 14. Mice were bled retroorbitally on day 31 and their sera were analyzed by MIF-specific ELISA.

C.ELISA

ELISA plates were coated with MIF-C1 at a concentration of 5. mu.g/ml. The ELISA plate was blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Preimmune sera of the same mice were also tested as controls. The results are shown in fig. 4C. Mouse antisera to MIF-C1 coupled to Q β capsid protein were significantly reactive, whereas preimmune sera did not react with MIF (fig. 4C, data not shown). In a dilution series of MIF-C1 antiserum conjugated to Q.beta.capsid protein, the maximum half titer (half-maximum titer) was reached at 1: 84000.

Figure 4C shows ELISA signals obtained from sera of mice vaccinated with MIF-C1 coupled to Q β capsid protein. Female Balb/c mice were subcutaneously vaccinated with 25 μ g of vaccine in PBS on days 0 and 14. anti-MIF-C1 serum IgG was measured on day 31. One of the mice was analyzed for pre-immune serum as a control. The results of the serum dilution are shown as optical density at 450 nm. All vaccinated mice developed high antibody titers. No MIF-specific antibodies were detected in the control.

Example 5

Coupling of rMIF-C1 with fr capsid protein and HBcAg-Lys-2cys-Mut capsid protein coupling of rMIF-C1 with fr capsid protein

Mu.l of a solution of 3.1mg/ml fr capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with 3. mu.l of 100mM SMPH stock solution (Pierce) dissolved in DMSO at 25 ℃ for 30 minutes. In a parallel reaction, the fr capsid protein is first alkylated with iodoacetamide and then reacted with SMPH under the same reaction conditions as described above. The reaction solution was subsequently dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. 80. mu.l and 1. mu.l of 36mM in H were dissolved 5.7mg/ml rMIF-C1 protein in 20mM Hepes, 150mM NaCl pH7.2₂A stock solution of TCEP (Pierce) for O was reacted at 25 ℃ for 1 hour. Mu.l derivatized dialyzed fr capsid protein and 50. mu.l derivatized alkylated dialyzed fr capsid protein were each reacted with 17. mu.l reduced rMIF-C1 for 2 hours at 25 ℃.

The coupling products were analyzed on a 16% SDS-PAGE gel (FIG. 5). Bands of expected sizes of 27kDa (rMIF-C1: apparent molecular weight 13kDa, apparent molecular weight of fr capsid protein 14kDa) and 29kDa (apparent molecular weight 13kDa of rMIF-C1, apparent molecular weight 15kDa of HBcAg-Lys-2 cys-Mut) could be detected in the coupling reaction, but were not detected in the fr capsid protein and rMIF-C1 solutions, which clearly confirmed the coupling.

Coupling of rMIF-C1 with hepatitis HBcAg-Lys-2cys-Mut capsid protein

Mu.l of a solution of 1.2mg/ml HBcAg-Lys-2cys-Mut capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was mixed with 1.4. mu.l of SMPH (Pierce) (from dissolution in100mM stock solution of DMSO) was reacted at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. 80. mu.l of a solution of 5.7mg/ml rMIF-C1 protein in 20mM Hepes, 150mM NaCl pH7.2 was mixed with 1. mu.l of TCEP (Pierce) (from H solution)₂O36 mM stock solution) was reacted at 25 ℃ for 1 hour. Then 60. mu.l of derivatized and dialyzed HBcAg-Lys-2cys-Mut capsid protein were reacted with 20. mu.l of reduced rMIF-C1 for 2 hours at 25 ℃.

The coupled products were analyzed on a 16% SDS-PAGE gel under reducing conditions (FIG. 5). An extra band of the expected size of approximately 28kDa (rMIF-C1: apparent molecular weight 13kDa, HBcAg-Lys-2 cys-Mut: apparent molecular weight 15kDa) could be detected in the coupling reaction, but not in the derivatized HBcAg-Lys-2cys-Mut or rMIF-C1 solution, clearly confirming the coupling.

The samples added to the gel of fig. 5 were as follows:

lane 1: and (4) molecular weight standard. And (2) a step: rMIF-C1 before coupling. And (3) a step: coupled rMIF-C1-fr capsid protein. And 4, a 4 th step: a derivatized fr capsid protein. And (5) a step: rMIF-C1-fr coupled to alkylated fr capsid protein. And 6, a step of: alkylated and derivatized fr capsid proteins. And 7, a step of: coupled rMIF-HBcAg-Lys-2 cys-Mut. Lanes 8 and 9: derivatized HBcAg-Lys-2 cys-Mut. The gel was stained with Coomassie Brilliant blue. Molecular weights of the standard proteins are shown in the left blank.

Example 6

A. Introduction of amino acid joint containing cysteine residue, expression and purification of mouse RANKL

A fragment recombinantly expressing nuclear factor kb ligand receptor activator (RANKL, also known as osteoclast differentiation factor, osteoprotegerin ligand and tumor necrosis factor-related activation-induced cytokine) having an N-terminal linker with one cysteine for coupling to VLPs.

Construction of expression plasmids

The C-terminal coding region of the RANKL gene was PCR amplified using oligonucleotides RANKL-UP and RANKL-DOWN. RANKL-UP contains an internal ApaI site and RANKL-DOWN contains an internal XhoI site. The PCR product was digested with ApaI and XhoI and ligated into pGEX-6p1(Amersham Pharmacia). The resulting plasmid was named pGEX-RANKL. All steps were performed according to standard molecular biology methods and the sequence was confirmed. Plasmid pGEX-RANKL encodes glutathione S-transferase-Prescission cleavage site-cysteine-containing amino acid linker-RANKL (GST-PS-C-RANKL) fusion protein. The cysteine-containing amino acid linker has the sequence GCGGG. The construct also contained a hexahistidine tail between the cysteine-containing amino acid linker and the RANKL sequence.

Oligonucleotide:

RANKL-UP：

5’-CTGCCAGGGGCCCGGGTGCGGCGGTGGCCATCATCACCACCATCACCAGCGCTTCTCAGGAG-3’(SEQ ID NO：316)

RANKL-DOWN：

5’-CCGCTCGAGTTAGTCTATGTCCTGAACTTTGAAAG-3’(SEQ ID NO：317)

the protein sequence of GST-PS-C-RANKL (SEQ ID NO: 318) and the cDNA sequence of GST-PS-C-RANKL (SEQ ID NO: 319)

1 M S P I L G Y W K I K G L V Q P T R L L L E Y L E

1 atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaa

26 E K Y E E H L Y E R D E G D K W R N K K F E L G L

76 gaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttg

51 E F P N L P Y Y I D G D V K L T Q S M A I I R Y I

151 gagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatata

76 A D K H N M L G G C P E E R A E I S M L E G A V L

226 gctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttg

101 D I R Y G V S R I A Y S K D F E T L K V D F L S K

301 gatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaag

126 L P E M L K M F E D R L C H K T Y L N G D H V T H

376 ctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccat

151 P D F M L Y D A L D V V L Y M D P M C L D A F P K

451 cctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaa

176 L V C F K K R I E A I P Q I D K Y L K S S K Y I A

526 ttagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagca

201 W P L Q G W Q A T F G G G D H P P K S D L E V L F

601 tggcctttgcagggctggcaagccacgtttgggggtggcgaccatcctccaaaatcggatctggaagttctgttc

226 Q G P G C G G G H H H H H H Q R F S G A P A H H E

676 cagGGGCCCGGGTGCGGCGGTGGCCATCATCACCACCATCACCAGCGCTTCTCAGGAGCTCCAGCTATGATGGAA

251 G S W L D V A Q R G K P E A Q P F A H L T T N A A

751 GGCTCATGGTTGGATGTGGCCCAGCGAGGCAAGTCTGAGGCCCAGCCATTTGCACACCTCACCATCAATGCTGCC

276 S I P S G S H R V T L S S W Y H D R G W A K I S N

826 AGCATCCCATCGGGTTCCCATAAAGTCACTCTGTCCTCTTGGTACCACGATCGAGGCTGGGCCAAGATCTCTAAC

301 M T L S N G K L R V N Q D G F Y Y L Y A N I C F R

901 ATGACGTTAAGCAACGGAAAACTAAGGGTTAACCAAGATGGCTTCTATTACCTGTACGCCAACATTTGCTTTCGG

326 H H E T S G S V P T D Y L Q L M V Y V V K T S I K

976 CATCATGAAACATCGGGAAGCGTACCTACAGACTATCTTCAGCTGATGGTGTATGTCGTTAAAACCAGCATCAAA

351 I P S S H N L M K G G S T K N W S G N S E F H F Y

1051 ATCCCAAGTTCTCATAACCTGATGAAAGGAGGGAGCACGAAAAACTGGTCGGGCAATTCTGAATTCCACTTTTAT

376 S I N V G G F F K L R A G E E I S I Q V S N P S L

1126 TCCATAAATGTTGGGGGATTTTTCAAGCTCCGAGCTGGTGAAGAAATTAGCATTCAGGTGTCCAACCCTTCCCTG

401 L D P D Q D A T Y F G A F K V Q D I D

l201 CTGGATCCGGATCAAGATGCGACGTACTTTGGGGCTTTCAAAGTTCAGGACATAGACTAACTCGAGCGG

Expression and purification of C-RANKL

Competent E.coli BL21(DE3) Gold pLys cells were transformed with plasmid pGEX-RANKL. Individual colonies on kanamycin-and chloramphenicol-containing agar plates were amplified in liquid medium (LB medium, 30. mu.g/ml kanamycin, 50. mu.g/ml chloramphenicol), and incubated overnight at 30 ℃ with shaking at 220 rpm. Then 1: 100v/v of the overnight broth was inoculated into 1LLB (containing 30. mu.g/ml kanamycin) and cultured at 24 ℃ until OD600 ═ 1. Expression was induced with 0.4mM IPTG. After 16 hours, the cells were collected by centrifugation at 5000 rpm. The cell pellet was suspended in lysis buffer (50mM Tris-HCl, pH 8; 25% sucrose; 1mM EDTA, 1% NaN)₃；10mMDTT；5mM MgCl₂(ii) a 1mg/ml lysozyme; 0.4U/ml DNAse) for 30 minutes. 2.5 volumes of buffer A (50mM Tris-HCl, pH 8.0; 1% Triton X100; 100mM NaCl; 0.1% NaN 3; 10mM DTT; 1mM PMSF) were added and incubated at 37 ℃ for 15 minutes. Cells were sonicated and pelleted at 9000rpm for 15 minutes. The supernatant was immediately subjected to GST-affinity chromatography.

5ml GST-Trap FF column (Amersham Pharmacia) was purified with PBS pH7.3(140mM NaCl, 2.7mM KCl, 10mM Na ₂HPO₄，1.8mM KH₂PO₄) And (4) balancing. The supernatant was applied to a 5ml GST-Trap FF column, followed by washing the column with 5 column volumes of PBS. The protein GST-PS-C-RANKL was eluted with 50mM Tris-HCl, pH 8.0, containing 10mM GSSH.

The purified GST-PS-C-RANKL protein was digested with the protease PreScission (Amersham pharmacia). Digestion was carried out at 37 ℃ for 1 hour with a molar ratio of GST-PS-C-RANKL to PreScission of 500/1.

In addition, the protease digestion reaction was buffer exchanged using a HiPrep26/10 desalting column (Amersham Pharmacia), the protein containing fractions were pooled and immediately subjected to another step of GST affinity chromatography using the same conditions as reported previously. The purification of C-RANKL was analyzed on SDS-PAGE gels under reducing conditions, as shown in FIG. 6. The molecular weight of the standard protein is shown in the left blank of the gel in the figure. The gel was stained with Coomassie Brilliant blue. Cleaved C-RANKL is present in the effluent (unbound fraction), while uncleaved GST-PS-C-RANKL, cleaved GST-PS and PreScission are bound to the column. C-RANKL protein with expected size of 22kDa is obtained in high purity.

The samples loaded onto the gel of fig. 6 were as follows:

lane 1: low molecular weight standards. Lanes 2 and 3: after 16 hours induction with 0.4mM IPTG, the empty vectors pGEX6p1 and pGEX-RANKL were used to transform the supernatant of the lysate of BL21/DE3 cells, respectively. And 4, a 4 th step: GST-PS-C-RANKL protein purified after passing through GST-Trap FF column. And (5) a step: fraction unbound with GST-Trap FF column. And 6, a step of: GST-PS-C-RANKL protein purified after cleavage by PreScission protease. And 7, a step of: unbound fraction of GST-TrapFF column loaded with GST-RANKL digest contains purified C-RANKL. And (8) a step: binding fractions of GST-Trap FF column loaded with GST-PS-C-RANKL digest and eluted with GSH.

Coupling of C-RANKL to Q beta capsid protein

A solution of 120. mu. M Q β capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock solution on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed reaction mixture of Q.beta.was reacted with a C-RANKL solution (final concentration: 60. mu. M Q. beta., 60. mu. M C-RANKL) for 4 hours at 25 ℃ on a shaker.

The coupled product was analyzed by SDS-PAGE.

C.C-RANKL conjugation to fr capsid proteins

mu.M fr capsid protein in 20mM Hepes, 150mM NaCl (pH7.2) was reacted with 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock solution on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed fr capsid protein reaction mixture was reacted with a C-RANKL solution (final concentration: 60. mu.M fr capsid protein, 60. mu. M C-RANKL) for 4 hours at 25 ℃ on a shaker. The coupled product was analyzed by SDS-PAGE.

Coupling of C-RANKL with HBcAg-Lys-2cys-Mut

A solution of 120. mu.M HBcAg-Lys-2cys-Mut capsid in 20mM Hepes, 150mM NaClpH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock in a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed HBcAg-Lys-2cys-Mut reaction mixture was reacted with C-RANKL solution (final concentration: 60. mu. MHBcAg-Lys-2cys-Mut, 60. mu. M C-RANKL) for 4 hours at 25 ℃ on a shaker.

The coupled product was analyzed by SDS-PAGE.

E.C-RANKL conjugation to pili

A solution of 125. mu.M E.coli type 1 pili in 20mM Hepes pH7.4 was reacted with a 50-fold molar excess of the cross-linking agent SMPH diluted with DMSO stock solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted by means of a PD-10 column (Amersham-Pharmacia Biotech). The protein-containing fractions eluted from the column were combined, and the desalted derivative pilin was reacted with a C-RANKL solution (final concentration: 60. mu.M pilus, 60. mu.MC-RANKL) for 4 hours at 25 ℃ on a shaker. The coupled product was analyzed by SDS-PAGE.

Example 7

A. Introduction of amino acid linker containing cysteine residue, expression and purification of truncated form of mouse prion protein

Recombinant expression of mouse prion protein (designated mPrP)_t) A truncated form of (amino acid 121-230) fused at the C-terminus to a GGGGCG amino acid linker for coupling to VLPs and pili. For ease of purification, the protein was fused to the N-terminus of a human Fc-fragment. To cleave the Fc portion of the fusion protein after purification, an Enterokinase (EK) cleavage site was introduced after the EK cleavage site.

mPrP_t-EK-Fc^*Construction of

From plasmid pBP^CMVPrP-Fc as template, mouse PrP was amplified with primers 5 'PrP-BamHI and 3' PrP-NheIPCR _t。pBP^CMVPrP-Fc contains the wild-type sequence of the mouse prion protein. 5 'PrP-BamHI contains an internal BamHI site and contains an ATG, 3' PrP-NheI contains an internal NheI site.

For the PCR reaction, 50. mu.l of the reaction mixture (1 unit PFX Platinum polymerase, 0.3mM dNTPs and 2mM MgSO)₄) 0.5. mu.g each of the primers and 200ng of the template DNA were used. The temperature cycle was as follows: 94 ℃ for 2 minutes, then 94 ℃ (15 seconds), 50 ℃ (30 seconds), 68 ℃ (45 seconds) for 5 cycles, then 94 ℃ (15 seconds), 64 ℃ (30 seconds), 68 ℃ (45 seconds) for 20 cycles, followed by 68 ℃ for 10 minutes.

The PCR product was digested with BamHI and NheI and inserted into pCEP-SP-EK-Fc containing GGGGCG linker sequence at the 5' end of EK cleavage sequence^*In (1). The resulting plasmid was designated pCEP-SP-mPrP_t-EK-Fc^*。

All other steps were performed according to standard molecular biology methods.

Oligonucleotide:

primer 5' PrP-BamHI

5’-CGG GAT CCC ACC ATG GTG GGG GGC CTT GG-3’(SEQ ID NO：321)

Primer 3' PrP-NheI

5’-CTA GCT AGC CTG GAT CTT CTC CCG-3’(SEQ ID NO：322)

mPrP_t-EK-Fc^*Expression and purification of

Using plasmid pCEP-SP-mPrP_t-EK-Fc^*293-EBNA cells (Invitrogen) were transfected and purified using a protein A-sepharose column as described in example 1.

mPrP_t-EK-Fc^*The protein sequence of SEQ ID NO: 323, to be listed therein.

Cleaved mPrP_tComprises the amino acid sequence shown in SEQ ID NO: 324, comprising a GGGGCG linker at its C-terminus.

Purified fusion protein mPrP_t-EK-Fc^*Cleaved with enterokinase and analyzed on a 16% SDS-PAGE gel under reducing conditions before and after enterokinase cleavage. The gel was stained with Coomassie Brilliant blue. The results are shown in fig. 7. The molecular weight of the standard protein is shown in the left blank of the gel in the figure. mPrP_t-EK-Fc^*The fusion protein was detected as a 50kDa band. Cleaved mPrP containing GGGGCG amino acid linker fused to its C-terminus_tThe protein was detected as a broad band of 18-25 kDa. mPrP_tThe identity of (c) was confirmed by Western blot (data not shown). Thus, mPrP having a C-terminal amino acid linker with a cysteine residue can be expressed and purified_tFor coupling to VLPs and pili.

The samples loaded onto the gel of fig. 7 were as follows:

lane 1: and (4) molecular weight standard. And (2) a step: mPrP before cleavage_t-EK-Fc^*. And (3) a step: cleaved mPrP_t。

B.mPrP_tCoupling to Q beta capsid

A solution of 120M Q β capsid in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock solution on a shaker at 25 ℃ for 30 minutes. The reaction solution was subsequently dialyzed at 4 ℃ against 1L of 20mM Hepes, 150mM NaCl pH7.22 times for 2 hours each. Dialyzed Q β reaction mixture and mrp _tSolution (final concentration: 60. mu. M Q. beta., 60. mu.M mPrP_t) The reaction was carried out on a shaker at 25 ℃ for 4 hours. The coupled product was analyzed by SDS-PAGE.

C.mPrP_tConjugation to fr capsid proteins

mu.M fr capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted from DMSO stock in a shaker at 25 ℃ for 30 min. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed fr reaction mixture and mPrP_tSolution (final concentration: 60. mu.M fr 60. mu.M mPrP_t) The reaction was carried out on a shaker at 25 ℃ for 4 hours. The coupled product was analyzed by SDS-PAGE.

D.mPrP_tCoupling with HBcAg-Lys-2cys-Mut

A solution of 120. mu.M HBcAg-Lys-2cys-Mut capsid in 20mM Hepes, 150mM NaClpH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock in a shaker at 25 ℃ for 30 minutes. The reaction solution was subsequently dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. Dialyzed HBcAg-Lys-2cys-Mut reaction mixture and mPrP_tSolution (final concentration: 60. mu. MHBcAg-Lys-2cys-Mut, 60. mu.M mPrP_t) The reaction was carried out on a shaker at 25 ℃ for 4 hours. The coupled product was analyzed by SDS-PAGE.

E.mPrP_tCoupling to pili

A solution of 125. mu.M E.coli type 1 pili in 20mM Hepes pH7.4 was reacted with a 50-fold molar excess of the cross-linking agent SMPH (Pierce) diluted from a DMSO stock solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted by means of a PD-10 column (Amersham-Pharmacia Biotech). Combining the protein-containing fractions eluted from the column, desalted derivative pilin and mPrP _tSolution (final concentration: 60. mu.M pilus, 60. mu.M mPrP_t) The reaction was carried out on a shaker at 25 ℃ for 4 hours.

The coupled product was analyzed by SDS-PAGE.

Example 8

A. Coupling of prion peptides to Q β capsid protein: prion peptide vaccine

The following prion peptides were chemically synthesized: CSAMSRPMIHFGNDWEDRYYRENMYR ("cprplong") and CGNDWEDRYYRENMYR ("cprpshrt"), which contain an added N-terminal cysteine residue for chemical coupling to Q β as described below, for coupling to VLPs and pili.

5ml of a solution of 140. mu. M Q β -capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 108. mu.l of a 65mM aqueous SMPH solution (Pierce) on a shaker at 25 ℃ for 30 minutes. The reaction solution was subsequently dialyzed against 5L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed reaction mixture was reacted with 1.35. mu.l of 2mM peptide cprppshort stock (dissolved in DMSO) (1: 2 peptide/Q.capsid ratio) or with 2.7. mu.l of the same stock (1: 1 peptide/Q.ratio). Mu.l of 10mM peptide cprplong stock solution (dissolved in DMSO) was reacted with 100. mu.l of the dialyzed reaction mixture. The coupling reaction was carried out overnight in a water bath at 15 ℃. The reaction mixture was then dialyzed against 2X 5L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 24 hours.

The coupled product was centrifuged and the supernatant and pellet were analyzed on a 16% SDS-PAGE gel under reducing conditions. The gel was stained with Coomassie Brilliant blue. The results are shown in fig. 16. The molecular weight of the standard protein is shown in the left blank of the gel in the figure. The covalent coupling of the peptides cprpshrrt and cprplong to the Q capsid protein is clearly confirmed by the band between 16.5 and 25kDa in molecular weight.

The samples loaded onto the gel of fig. 16A were as follows:

lane 1: purified Q capsid protein. And (2) a step: the pre-coupled derivatized Q β capsid protein. Lanes 3-6: q β capsid protein-cprpshrrt couplings were performed at 1: 2 peptide/Q.ratio (lanes 3, 4) and 1: 1 peptide/Q.ratio (lanes 5, 6). Soluble fractions (lanes 3, 5) and insoluble fractions (lanes 4, 6) are shown.

The samples loaded onto the gel of fig. 16B were as follows:

lane 1: and (4) molecular weight standard. And (2) a step: the pre-coupled derivatized Q β capsid protein. Lanes 3 and 4: q β capsid protein-cprplong conjugate reactions. Soluble fraction (lane 3) and insoluble fraction (lane 4) are shown.

B. Coupling of prion peptides to fr capsid proteins

A solution of 120. mu.M fr capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 10-fold molar excess of SMPH (Pierce) diluted from DMSO stock in a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed fr reaction mixture was reacted overnight with equimolar concentrations of the peptide cprpshrrt or 1: 2 cprpplong/fr on a shaker at 16 ℃. The coupled product was analyzed by SDS-PAGE.

C. Coupling of prion peptides to HBcAg-Lys-2cys-Mut

mu.M HBcAg-Lys-2cys-Mut in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 10-fold molar excess of SMPH (Pierce) diluted from DMSO stock for 30 min at 25 ℃ in a shaker. The reaction solution was subsequently dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed HBcAg-Lys-2cys-Mut reaction mixture was reacted overnight with equimolar concentrations of the peptide cprpshrrt or 1: 2 cplong/HBcAg-Lys-2 cys-Mut on a shaker at 16 ℃. The coupled product was analyzed by SDS-PAGE.

D. Coupling of prion peptides to pili

A solution of 125. mu.M E.coli type 1 pili in 20mM Hepes pH7.4 was reacted with a 50-fold molar excess of the cross-linking agent SMPH (Pierce) diluted from a DMSO stock solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted by means of a PD-10 column (Amersham-Pharmacia Biotech). The protein-containing fractions eluted from the column were pooled, and the desalted derivatized pilin was reacted with prion peptide equimolar or in a 1: 2 peptide/pilus ratio on a shaker at 16 ℃ overnight. The coupled product was analyzed by SDS-PAGE.

Example 9

Cloning, expression and purification of IL-13 coupled to VLPs and pili

A. Cloning and expression of Interleukin 13(IL-13) to couple VLP and pili, it contains a cysteine residue at the N-terminal amino acid linker

a) Cloning of mouse IL-13 (HEK-293T) for expression as Fc fusion protein in mammalian cells

DNA for cloning of IL-13 was isolated by RT-PCR from in vitro activated splenocytes obtained as follows: CD4+ T cells were isolated from mouse spleen cells and incubated in IMDM (+ 5% FCS +10ng/ml IL4) in 6-well plates previously coated with anti-CD 3 and anti-CD 28 antibodies for 3 days. IL13 was amplified by one-step RT-PCR (Qiagen one-step PCR kit) using RNA from these cells. Reverse transcription of RNA Using primer XhoIL13-R, PCR amplification of IL13 cDNA Using primers NheIL13-F (SEQ ID NO: 338) and XhoIL13-R (SEQ ID NO: 339). The amplified IL13 cDNA was ligated into the pMOD vector (resulting in the vector pMODB1-IL13) using NheI/XhoI restriction sites. pMODB1-IL13 was digested with BamHI/XhoI, and the IL-13-containing fragment was ligated to pCEP-SP-XA-Fc previously digested with BamHI/XhoI^*(Δ xho) vector, which was ligated with pCEP-SP-XA-Fc^*Similar in structure but with one XhoI site removed at the end of the Fc sequence. The plasmid generated after ligation (pCEP-SP-IL13-Fc) was sequenced and used to transfect HEK-293T cells. The plasmid encodes an IL13 construct fused to the amino acid sequence ADPGCGGGGGLA at the N-terminus of the mature IL-13 sequence. This sequence comprises the amino acid linker sequence GCGGGGG flanked by additional amino acids introduced during cloning. IL13-Fc was purified from the supernatant of pCEP-SP-IL13-Fc transfected cells using protein A resin. The expression results are shown in FIG. 17B (see example 10 for a sample). Cleavage of the fusion protein with factor Xa releases the mature IL-13 fused N-terminally to the above amino acid sequence, obtaining a protein, hereinafter referred to as "mouse C-IL-13-F", having the amino acid sequence shown in SEQ ID NO: 328. The results of FIG. 17B clearly confirm the expression of the IL-13 construct.

b) Cloning of mouse IL-13 (HEK-293T) for expression in mammalian cells fused at the N-terminus to GST (glutathione-S-transferase)

cDNA for cloning of IL-13 containing GST at the N-terminus was obtained as described in (a) above for T cells activated by TH 2. IL-13 was amplified from this cDNA using primers Nhelink1IL13-F and IL13 StopXhoNot-R. The PCR product was digested with NheI and XhoI and ligated into pCEP-SP-GST-EK vector previously digested with NheI/XhoI. HEK-293T cells were transfected with a plasmid (pCEP-SP-GST-IL13) that could be isolated from the ligation reaction. The resulting IL13 construct encoded by this plasmid fused the amino acid sequence LACGGGGG N-terminal to the mature IL-13 sequence. This sequence comprises the amino acid linker sequence ACGGGGG flanked by additional amino acids introduced during cloning. The culture supernatant of cells transfected with pCEP-SP-GST-IL13 contained the fusion protein GST-IL13, which was purified by glutathione affinity chromatography according to standard procedures. Cleavage of the fusion protein with enterokinase released mature IL-13 fused at the N-terminus to the above amino acid sequence to obtain a protein, hereinafter referred to as "mouse C-IL-13-S", having the amino acid sequence of SEQ ID NO: 329 of the sequence listing.

B. Coupling of mouse C-IL-13-F, mouse C-IL-13-S and Q beta capsid protein

A solution of 120. mu. M Q β capsid in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted from DMSO stock on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed Q.beta.reaction mixture was reacted with a mouse C-IL-13-F or mouse C-IL-13-S solution (final concentration: 60. mu. M Q. beta. capsid protein, 60. mu.M mouse C-IL-13-F or mouse C-IL-13-S) on a shaker at 25 ℃ for 4 hours. The coupled product was analyzed by SDS-PAGE.

C. Coupling of mouse C-IL-13-F, mouse C-IL-13-S to fr capsid protein

mu.M fr capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted from DMSO stock in a shaker at 25 ℃ for 30 min. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed fr capsid protein reaction mixture was reacted with a mouse C-IL-13-F or mouse C-IL-13-S solution (final concentration: 60. mu.M fr capsid protein, 60. mu.M mouse C-IL-13-F or mouse C-IL-13-S) for 4 hours at 25 ℃ on a shaker. The coupled product was analyzed by SDS-PAGE.

D. Coupling of mouse C-IL-13-F or mouse C-IL-13-S solutions with HBcAg-Lys-2cys-Mut

A solution of 120. mu.M HBcAg-Lys-2cys-Mut capsid in 20mM Hepes, 150mM NaClpH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock in a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed HBcAg-Lys-2cys-Mut reaction mixture was reacted with mouse C-IL-13-F or mouse C-IL-13-S solution (final concentration: 60. mu.M HBcAg-Lys-2cys-Mut, 60. mu.M mouse C-IL-13-F or mouse C-IL-13-S) on a shaker at 25 ℃ for 4 hours. The coupled product was analyzed by SDS-PAGE.

E. Coupling of mouse C-IL-13-F or mouse C-IL-13-S solutions to pili

A solution of 125. mu.M E.coli type 1 pili in 20mM Hepes pH7.4 was reacted with a 50-fold molar excess of the cross-linking agent SMPH (Pierce) diluted from a DMSO stock solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted by means of a PD-10 column (Amersham-Pharmacia Biotech). The protein-containing fractions eluted from the column were pooled, and the desalted derivative pilin was reacted with a mouse C-IL-13-F or mouse C-IL-13-S solution (final concentration: 60. mu.M pilus, 60. mu.M mouse C-IL-13-F or mouse C-IL-13-S) for 4 hours at 25 ℃ on a shaker. The coupled product was analyzed by SDS-PAGE.

Example 10

Cloning and expression of Interleukin 5(IL-5) containing an N-terminal amino acid linker containing cysteine residues for conjugation to VLPs and pili

Cloning of IL-5 for expression as inclusion bodies in E.coli

IL-5 was PCR amplified from an ATCC clone (pmIL 5-4G; ATCC No. 37562) using the following two primers: spelinker3-F1(SEQ ID NO: 340) and IL5StopXho-R (SEQ ID NO: 342). A second PCR was performed using this PCR product as a template, using primers SpeNlinker3-F2(SEQ ID NO: 341) and IL5 StopXho-R. The insert was digested with SpeI and NotI. This insert was ligated into pET vector derivatives (pMODEC3-8 vector) previously digested with NheI and NotI (unphosphorylated), and E.coli TG1 cells were transformed. Cloning into the pMODEC3-8 vector produced an IL5 construct containing a hexa-histidine tail at its N-terminus, followed by an enterokinase site, an N-terminal γ 3 amino acid linker containing cysteine residues (flanked on the C-terminus by the sequence AS and on the N-terminus by the sequence ALV), and the mature form of the IL5 gene. The protein released by enterokinase cleavage was called "mouse C-IL-5-E" (SEQ ID NO: 332). The sequence of the resulting plasmid DNA of clone pMODC6-IL5.2 (also known as pMO DC6-IL5) was confirmed by DNA sequencing, and E.coli strain BL21 was transformed therewith.

Clone pMODC6-IL5/BL21 was grown overnight in 5ml LB containing 1mg/L ampicillin. 2ml of the culture was diluted with 100ml of a culture (TB) containing 1mg/L ampicillin. When the culture reached an optical density OD600 of 0.7, 0.1ml of 1M isopropyl β -D-thiogalactopyranoside (IPTG) solution was added to induce culture. Samples of 10ml were taken every 2 hours. The samples were centrifuged at 4000 Xg for 10 min. The pellet was resuspended in 0.5ml lysis buffer (pH8) containing 50mM Tris-HCl, 2mM EDTA, 0.1% triton X-100. After adding 20. mu.l lysozyme (40mg/ml) and incubating the tubes at 4 ℃ for 30 minutes, the cells were sonicated for 2 minutes. Add 100. mu.l 50mM MgCl₂Solution and 1ml benzonase. Cells were then incubated at room temperature for 30 minutes and centrifuged at 13000 Xg for 15 minutes.

The supernatant was discarded and the pellet was boiled in 100. mu.l SDS loading buffer at 98 ℃ for 5 minutes. A10. mu.l sample in loading buffer was analyzed by SDS-PAGE under reducing conditions (FIG. 17A). The gel of FIG. 17A clearly demonstrates the expression of the IL-5 construct. The samples loaded onto the gel of fig. 17A were as follows:

and (3) M times: standard molecular weight protein (NEB, broad range pre-stained standard protein). Lane 1: cell extracts of the first 1ml of culture were induced. And (2) a step: cell extracts of 1ml culture medium after 4 hours of induction.

Cloning of IL-5 for expression in mammalian cells (HEK-293T)

a) IL-5 fused at the N-terminus to an amino acid linker containing a cysteine residue and at the C-terminus to an Fc fragment

The following constructs were cloned using the template described in (A) (ATCC clone 37562). Plasmid pMODB1-IL5 (a pET derivative) was digested with BamHI/XhoI to generate a small fragment encoding IL5 fused to an N-terminal amino acid linker containing cysteine. This fragment was ligated into vector pCEP-SP-XA-Fc previously digested with BamHI and XhoI^*(Δ Xho). The ligation was electroporated into E.coli TG1 strain, and the plasmid DNA of the resulting clone pCEP-SP-IL5-Fc.2 was sequenced to confirm its sequence and used to transfect HEK-293T cells. The plasmid-encoded IL5 construct contained amino acid sequence ADPGCGGGGGLA fused N-terminal to the mature IL-5 sequence. This sequence comprises the amino acid linker sequence GCGGGGG, which contains one cysteine flanked by other amino acids introduced during cloning. The IL-5 protein released by cleavage of the fusion protein with factor-Xa is hereinafter referred to as "mouse C-IL-5-F" (SEQ ID NO: 333).

After transfection and selection on puromycin, culture supernatants were analyzed by Western blot using anti-His (mouse) and anti-mouse IgG antibodies conjugated to horseradish peroxidase (fig. 17B). Anti-mouse IgG antibodies conjugated to horseradish peroxidase also detected Fc-fusion proteins. Protein purification was performed by affinity chromatography using protein a resin. The results in FIG. 17B clearly confirm the expression of the IL-5 construct.

The samples loaded on the Western blot of fig. 17B were as follows:

lane 1: HEK culture supernatant (20. mu.l) expressing IL 5-Fc. SDS-PAGE was performed under reducing conditions. And (2) a step: HEK culture supernatant (20. mu.l) expressing IL 13-Fc. SDS-PAGE was performed under non-reducing conditions. And (3) a step: HEK culture supernatant (20. mu.l) expressing IL 5-Fc. SDS-PAGE was performed under non-reducing conditions.

b) Cloned IL-5 containing GST (glutathione-S-transferase) and an amino acid linker containing a cysteine residue fused at the N-terminus

IL-5(ATCC37562) was amplified with primers Nhe-link1-IL13-F and IL5 StopXho-R. After digestion with NheI and XhoI, the insert was ligated into pCEP-SP-GST-EK previously digested with NheI and XhoI. The resulting plasmid pCEP-SP-GST-IL5 was sequenced and used to transfect HEK-293T cells. The resulting plasmid-encoded IL-5 construct contains the amino acid sequence LACGGGGG fused to the N-terminus of the mature IL-5 sequence. This sequence comprises the amino acid linker sequence ACGGGGG, which contains one cysteine residue flanked by other amino acids introduced during cloning. The protein released by enterokinase cleavage is hereinafter referred to as "mouse C-IL-5-S" (SEQ ID NO: 334). The resulting protein was purified by affinity chromatography using glutathione affinity resin.

C. Coupling of mouse C-IL-5-F or mouse C-IL-5-S to Q beta capsid protein

A solution of 120. mu. M Q β capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted from DMSO stock in a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed Q.beta.reaction mixture was reacted with a mouse C-IL-5-F or mouse C-IL-5-S solution (final concentration: 60. mu. M Q. beta. capsid protein, 60. mu.M mouse C-IL-5-F or mouse C-IL-5-S) for 4 hours at 25 ℃ on a shaker. The coupled product was analyzed by SDS-PAGE.

D. Conjugation of mouse C-IL-5-F or mouse C-IL-5-S to fr capsid proteins

mu.M fr capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted from DMSO stock in a shaker at 25 ℃ for 30 min. The reaction solution was then dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed fr reaction mixture was reacted with a mouse C-IL-5-F or mouse C-IL-5-S solution (final concentration: 60. mu. Mfr capsid protein, 60. mu.M mouse C-IL-5-F or mouse C-IL-5-S) for 4 hours at 25 ℃ on a shaker. The coupled product was analyzed by SDS-PAGE.

E. Coupling of mouse C-IL-5-F or mouse C-IL-5-S solutions with HBcAg-Lys-2cys-Mut

A solution of 120. mu.M HBcAg-Lys-2cys-Mut capsid in 20mM Hepes, 150mM NaClpH7.2 was reacted with a 25-fold molar excess of SMPH (Pierce) diluted with DMSO stock in a shaker at 25 ℃ for 30 minutes. The reaction solution was subsequently dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed HBcAg-Lys-2cys-Mut reaction mixture was reacted with mouse C-IL-5-F or mouse C-IL-5-S solution (final concentration: 60. mu.M HBcAg-Lys-2cys-Mut, 60. mu.M mouse C-IL-5-F or mouse C-IL-5-S) on a shaker at 25 ℃ for 4 hours. The coupled product was analyzed by SDS-PAGE.

F. Coupling of mouse C-IL-5-F or mouse C-IL-5-S solutions to pili

A solution of 125. mu.M E.coli type 1 pili in 20mM Hepes pH7.4 was reacted with a 50-fold molar excess of the cross-linking agent SMPH (Pierce) diluted from a DMSO stock solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted by means of a PD-10 column (Amersham-Pharmacia Biotech). The protein-containing fractions eluted from the column were pooled, and the desalted derivative pilin was reacted with a mouse C-IL-5-F or mouse C-IL-5-S solution (final concentration: 60. mu.M pilus, 60. mu.M mouse C-IL-5-F or mouse C-IL-5-S) for 4 hours at 25 ℃ on a shaker. The coupled product was analyzed by SDS-PAGE.

Example 11

Introduction of amino acid linker containing cysteine residue, expression, purification and coupling of mouse vascular endothelial growth factor-2 (mVEGFR-2, FLK1) fragment

The construct of murine vascular endothelial growth factor-2 (mVEGFR-2, FLK1) comprising the second and third ectodomains is recombinantly expressed as an Fc-fusion protein comprising, at the C-terminus, an amino acid linker containing a cysteine residue for coupling to VLPs and fimbriae. mVEGFR-2(2-3) The protein sequence of (2) was translated from the cDNA sequence of mouse FLK-1 (Matthews et al, Proc. Natl. Acad. Sci. USA88: 9026-9030(1991): accession number X59397; ig-like C2 type domain 2: amino acid 143-; ig-like C2 type domain 3: amino acids 241-. The mVEGFR-2(2-3) construct comprises the sequence of mVEGFR-2 from proline 126 to lysine 329 (numbering of the precursor protein). This construct contains, in addition to immunoglobulin-like C2 type domains 2 and 3, flanking regions in the mVEGFR-2 sequence before domain 2 and after domain 3 for the addition of an amino acid spacer. Amino acid linkers containing one cysteine residue were cloned into pCEP-SP-EK-Fc^*The vector (example 1) is fused to the C-terminus of the mVEGFR-2 sequence. Cloned into pCEP-SP-EK-Fc^*A fragment of mVEGFR-2 within the vector encodes the following amino acid sequence (SEQ ID NO: 345): PFIAS VSDQHGIVYI TENKNKTVVI PCRGSISNLN VSLCARYPEKRFVPDGNRIS WDSEIGFTLP SYMISYAGMV FCEAKINDET YQSIMYIVVVVGYRIYDVIL SPPHEIELSA GEKLVLNCTA RTELNVGLDF TWHSPPSKSHHKKIVNRDVK PFPGTVAKMF LSTLTIESVT KSDQGEYTCVASSGRMIKRN RTFVRVHTKP

Expression of recombinant mVEGFR-2(2-3) in eukaryotic cells

Using pCEP-SP-EK-Fc^*The vector expresses recombinant mVEGFR-2(2-3) in EBNA293 cells. pCEP-SP-EK-Fc^*The vector contains a BamHI and an NheI site encoding an amino acid linker containing a cysteine residue, an enterokinase cleavage site and a C-terminal human Fc region. mVEGFR-2(2-3) was PCR amplified from mouse 7-day embryo cDNA (Marathon-Ready cDNA, Clontech) with primer pair BamHI-FLK1-F and NheI-FLK 1-B. For the PCR reaction, 10pmol each of the primers and 0.5ng of cDNA (mouse 7-day embryo cDNA, Marathon-Ready cDNA, Clontech) were used in a 50.1 reaction mix (1.1 Advantage2 polymerase mix (50X), 0.2mM dNTPs and 5.110 × cDNA PCR reaction buffer). The temperature cycle was as follows: 94 ℃ for 1 min, 94 ℃ for 30 sec, 54 ℃ for 30 sec, 72 ℃ for 1 min for 5 cycles, followed by 94 ℃ (30 sec), 54 ℃ (30 sec), 70 ℃ (1 min) for 5 cycles, followed by 94 ℃ (20 sec), 54 ℃ (30 sec), 68 ℃ (1 min) for 30 cycles. The PCR product was digested with BamHI and NheI, inserted into pCEP-SP-EK-Fc digested with the same enzymes^*CarrierIn (1). The resulting plasmid was designated mVEGFR-2(2-3) -pCep-EK-Fc. All other steps were performed according to standard molecular biology methods.

Oligonucleotide:

1. primer BamHI-FLK1-F

5’-CGCGGATCCATTCATCGCCTCTGTC-3’(SEQ ID NO：343)

2. Primer NheI-FLK1-B

5’-CTAGCTAGCTTTGTGTGAACTCGGAC-3 ’(SEQ ID NO：344)

Transfection and expression of recombinant mVEGFR-2(2-3)

EBNA 293 cells were transfected with the mVEGFR-2(2-3) -pCep-EK-Fc construct described above and the cell serum-free supernatant was collected for purification as described in example 1.

Purification of recombinant mVEGFR-2(2-3)

Protein A purification of the expressed Fc-EK-mVEGFR-2(2-3) protein was performed as described in example 1. Subsequently, after the fusion protein was bound to protein A, mVEGFR-2(2-3) was cleaved from the Fc portion bound to protein A using enterokinase (enterokinase Max, Invitrogen). Digestion was carried out overnight at 37 ℃ (2.5 units enterokinase/100 μ l fusion protein-binding protein a beads). The released VEGFR-2(2-3) was separated from the Fc part still bound to protein A by brief centrifugation through a chromatography column (Micro Bio Spin, Biorad). To remove enterokinase, the effluent was treated with enterokinase away (invitrogen) according to the instructions.

Example 12

Coupling of mouse VEGFR-2 peptide with Q beta capsid protein, HbcAg-lys-2cys-Mut with pili, immunization of mice with VLP-peptide and pili-peptide vaccines

A. Conjugation of murine VEGFR-2 peptides to VLPs and pili

The following peptides were chemically synthesized (synthesized by Eurogenetic, belgium): murine VEGFR-2 peptide CTART ELNVGLDFTWHSPPSKSHHKK, used for chemical coupling to pili as described below.

Coupling of murine VEGFR-2 peptide to pili:a solution of 1mg/ml pilin 20mM hepespH7.4 in 1400. mu.l was reacted with 85. mu.l of 100mM aqueous Sulfo-MBS (Pierce) solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted through a PD-10 column (Amersham-pharmacia Biotech) and the protein-containing fractions eluted from the column (approximately 1.4mg protein) were pooled and reacted with a 2.5-fold molar excess (final volume) of murine VEGFR-2 peptide. For example, to 200. mu.l of an eluate containing about 0.2mg of the derivatized pilus, 2.4. mu.l of a 10mM peptide solution (dissolved in DMSO) is added. The mixture was incubated on a shaker at 25 ℃ for 4 hours and then dialyzed overnight at 4 ℃ against 2L of 20mM hepes pH 7.2. The coupling results were analyzed by SDS-PAGE under reducing conditions and are shown in FIG. 18A. The supernatant (S) and pellet (P) of each sample were loaded onto the gel along with pili and Sulfo-MBS crosslinker (Pierce) -derived pili. The samples loaded onto the gel of fig. 18A were as follows:

lane 1: a standard protein; lanes 2-5: coupled samples (pilus mouse: pilus coupled to murine peptide; pilus human: pilus coupled to human peptide); and 6, a step of: pili derived with Sulfo-MBS crosslinker; lanes 7-9: three fractions of the PD-10 column eluate. Fraction 2 is the peak fraction, and fractions 1 and 3 are fractions collected at the peak edge. The coupling band was clearly visible on the gel, confirming successful coupling of murine VEGFR-2 to the pili.

Coupling of murine VEGFR-2 peptide to Q β capsid protein:1ml of a solution of 1mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 20. mu.l of 100mM Sulfo-MBS (Pierce) in water on a shaker at room temperature for 45 minutes. The reaction solution was then dialyzed twice against 2L of 20mM Hepes pH7.4 at 4 ℃ for 2 hours each. Mu.l of the dialyzed reaction mixture was reacted with 12. mu.l of a 10mM peptide solution (dissolved in DMSO) on a shaker at 25 ℃ for 4 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes pH7.4 at 4 ℃ for 2X 2 hours. The coupling results were analyzed by SDS-PAGE under reducing conditions and are shown in FIG. 18B. The supernatant (S) of each sample was loaded onto the gel along with Q.beta.capsid protein and Sulfo-MBS crosslinker (Pierce) -derived Q.beta.capsid protein. The coupling was performed in duplicate. The following are providedThe samples were loaded onto the gel:

lane 1: a standard protein; lanes 2 and 5: q β capsid protein; lanes 3 and 6: q β capsid protein derivatized with Su1fo-MBS crosslinker; lanes 4 and 7: q β capsid protein coupled to murine VEGFR-2 peptide. The coupling band was clearly visible on the gel, confirming successful coupling of murine VEGFR-2 to the Q β capsid protein.

Coupling of murine VEGFR-2 peptide to HbcAg-lys-2 cys-Mut: A solution of 0.9mg/ml cysteine-free HbcAg capsid protein (example 31) in PBS pH7.4 (3 ml) was reacted with 37.5. mu.l of 100mM Sulfo-MBS (Pierce) in water for 45 minutes at room temperature on a shaker. The reaction solution was then dialyzed overnight against 2L of 20mM Hepes pH 7.4. After buffer exchange, the reaction solution was dialyzed against the same buffer for another 2 hours. The dialyzed reaction mixture was reacted with 3. mu.l of 10mM peptide solution (dissolved in DMSO) on a shaker at 25 ℃ for 4 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 2L of 20mM Hepes pH7.4, then the buffer was changed and dialyzed against the same buffer for an additional 2 hours. The coupling results were analyzed by SDS-PAGE under reducing conditions and are shown in FIG. 18C. The supernatant (S) of each sample was loaded onto the gel along with HbcAg-lys-2cys-Mut protein and Sulfo-MBS crosslinker-derived HbcAg-lys-2cys-Mut protein. The coupling was performed in duplicate. The coupling reaction was carried out in 2.5-fold and 10-fold molar excess of peptide. The following samples were loaded onto the gel:

lane 1: a standard protein; lanes 2, 4, 6, 8: supernatant (S) and pellet (P) of the coupling reaction with a 10-fold molar excess of peptide; lanes 3, 5, 7, 9: supernatant (S) and pellet (P) of the coupling reaction with 2.5-fold molar excess of peptide; and (6) a 10 th step: Sulfo-MBS-derived HbcAg-lys-2 cys-Mut; and (6) a 11 th step: HbcAg-lys-2 cys-Mut.

The coupling band was clearly visible on the gel, confirming successful coupling of murine VEGFR-2 to HbcAg-lys-2cys-Mut protein.

B. Immunization of mice:

pilus-peptide vaccine:

female C3H-HeJ (lacking Toll-like receptor 4) and C3H-HeN (wild-type) mice were inoculated with murine VEGFR-2 skin coupled to pilin without adjuvant. Approximately 100. mu.g of total protein from each sample was diluted to 200. mu.l with PBS and injected subcutaneously on days 0, 14, and 28. Mice were bled retroorbitally on days 14, 28, and 42, and sera on day 42 were analyzed by human VEGFR-2 specific ELISA.

Q β capsid protein-peptide vaccine:

female Black6 mice were inoculated with murine VEGFR-2 peptide coupled to Qp capsid protein with and without adjuvant (aluminum hydroxide). Approximately 100. mu.g of total protein from each sample was diluted to 200. mu.l with PBS and injected subcutaneously on days 0, 14, and 28. Mice were bled retroorbitally on days 14, 28, and 42, and sera on day 42 were analyzed by human VEGFR-2 specific ELISA.

HbcAg-lys-2cys-Mut vaccine:

female Black6 mice were inoculated with murine VEGGFR-2 peptide coupled to HbcAg-lys-2cys-Mut protein with and without adjuvant (aluminum hydroxide). Approximately 100. mu.g of total protein from each sample was diluted to 200. mu.l with PBS and injected subcutaneously on days 0, 14, and 28. Mice were bled retroorbitally on days 14, 28, and 42, and sera on day 42 were analyzed by human VEGFR-2 specific ELISA.

C.ELISA

Sera from immunized mice were assayed by ELISA using the immobilized murine VEGFR-2 peptide. The murine VEGFR-2 peptide was coupled to bovine RNAseA using the chemical cross-linker Sulfb-SPDP. ELISA plates were coated with conjugated RNAseA at a concentration of 10. mu.g/ml. The ELISA plate was blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Preimmune sera of the same mice were also tested as controls. Control ELISA experiments were performed with sera from mice immunized with unconjugated carrier and showed that the antibodies detected were specific for the respective peptides. The results are shown in FIGS. 4-6.

Pilus-peptide vaccine:

the results of ELISA are shown in FIG. 18D. The results for the serum dilutions are shown as optical density at 450 nm. The mean (including standard deviation) of the three mice is shown. All mice vaccinated produced IgG antibody titers against the murine VEGFR-2 peptide. No differences were seen between mice lacking Toll-like receptor 4 and wild-type mice, confirming the immunogenicity of the autoantigenic murine VEGFR-2 peptide in mice when coupled to pili. The vaccines injected into the mice were named after the corresponding sera analyzed.

Q β capsid protein-peptide vaccine:

the results for the serum dilutions are shown in fig. 18E as optical density at 450 nm. The mean (including standard deviation) of both mice is shown. All mice vaccinated produced IgG antibody titers against the murine VEGFR-2 peptide, confirming the immunogenicity of the autoantigen murine VEGFR-2 peptide in mice when coupled to the Q β capsid protein. The vaccines injected into the mice were named after the corresponding sera analyzed.

HbcAg-lys-2cys-Mut vaccine:

the results for the serum dilutions are shown in fig. 18F as optical density at 450 nm. The mean (including standard deviation) of the three mice is shown. All mice vaccinated produced IgG antibody titers against the murine VEGFR-2 peptide, confirming the immunogenicity of the autoantigen murine VEGFR-2 peptide in mice when coupled to the Q β capsid protein. The vaccines injected into the mice were named after the corresponding sera analyzed.

Example 13

Coupling of Abeta 1-15 peptide to HBcAg-lys-2cys-Mut and fr capsid proteins

The following a β peptide (DAEFRHDSGYEVHHQGGC) was chemically synthesized, which peptide comprises the amino acid sequence of human a β residues 1-15, fused at its C-terminus to the sequence GGC for coupling to VLPs and pili.

A.a) coupling of Abeta 1-15 peptide to HBcAg-lys-2cys-Mut with the aid of a crosslinking agent SMPH

833.3. mu.l of a solution of 1.2mg/ml HBcAg-lys-2cys-Mut protein in 20mM Hepes, 150mM NaCl pH7.4 were reacted with 17. mu.l of a 65mM SMPH (Pierce) aqueous solution on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed twice against 1L of 20mM Hepes, 150mM NaCl pH7.4 for 2 hours at 4 ℃ in a dialysis tube with a molecular weight of 10000 Da. 833.3 μ l of the dialyzed reaction mixture was reacted with 7.1 μ l of 50mM peptide stock solution (peptide stock solution dissolved in DMSO) on a shaker at 15 ℃ for 2 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 1L of 20mM Hepes, 150mM NaCl pH 7.4. The samples were aliquoted and frozen in liquid nitrogen and stored at-80 ℃ until used to immunize mice.

b) Coupling of Abeta 1-15 peptides to fr capsid protein Using Cross-linker SMPH

Mu.l of a solution of 2mg/ml fr capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 23. mu.l of a 65mM aqueous SMPH (Pierce) solution for 30 minutes at 25 ℃ on a shaker. The reaction solution was then dialyzed twice against 1L of 20mM Hepes, 150mM NaCl pH7.4 for 2 hours at 4 ℃ in a dialysis tube with a molecular weight of 10000 Da. Mu.l of the dialyzed reaction mixture was reacted with 5.7. mu.l of 50mM peptide stock solution (peptide stock solution dissolved in DMSO) on a shaker at 15 ℃ for 2 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 1L of 20mM Hepes, 150mM NaCl pH 7.4. The samples were aliquoted and frozen in liquid nitrogen and stored at-80 ℃ until used to immunize mice. Samples of the coupling reaction were analysed by SDS-PAGE under reducing conditions.

The results of the coupling experiments were analyzed by SDS-PAGE and are shown in FIG. 19A. The band of coupling corresponding to coupling of A.beta.1-15 to the fr capsid protein or to HBc-Ag-lys-2cys-Mut is clearly visible on the gel, indicated by the arrows in the figure, confirming successful coupling of A.beta.1-15 to the fr capsid protein and to the HBc-Ag-lys-2cys-Mut capsid protein. A beta 1-15 is coupled with fr capsid protein to form a plurality of coupling bands, while coupling with HBc-Ag-lys-2cys-Mut to form a coupling band mainly.

The following samples were loaded onto the gel of fig. 19A.

1: protein standards (molecular weight standard bands on kDa standard 7708S BioLabs. gel: 175, 83, 62, 47.5, 32.5, 25, 16.5, 6.5kDa from top to bottom). 2: derivatized HBc-Ag-lys-2 cys-Mut. 3: HBc-Ag-lys-2cys-Mut coupled to Abeta 1-15, supernatant of the sample taken at the end of the coupling reaction and centrifuged. 4: HBc-Ag-lys-2cys-Mut coupled to A.beta.1-15, a pellet of the sample taken and centrifuged at the end of the coupling reaction. 5: a derivatized fr capsid protein. 6: fr capsid protein coupled to A β 1-15, supernatant of the sample taken at the end of the coupling reaction and centrifuged. 7: fr capsid protein coupled to A β 1-15, pellet of sample taken and centrifuged at the end of the coupling reaction.

Immunization of Balb/c mice

Female Balb/c mice were inoculated subcutaneously twice on days 0 and 14 with 10. mu.g of Fr capsid protein coupled to A.beta.1-15 (Fr-A.beta.1-15) or 10. mu.g of HBc-Ag-lys-2cys-Mut (HBc-A.beta.1-15) coupled to A.beta.1-15 diluted in sterile PBS. Mice were bled retro-orbitally on day 22 and sera were analyzed by A β -1-15 specific ELISA.

C.ELISA

Coupling of Abeta 1-15 to bovine RNAseA using the chemical crosslinker Sulfo-SPDP. ELISA plates were coated with A.beta.1-15-RNAse conjugate at a concentration of 10. mu.g/ml. The ELISA plate was blocked and then incubated with serial dilutions of serum samples. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Sera from non-immunized mice were also tested as controls.

FIG. 19B shows ELISA signals obtained on day 22 using sera from mice immunized with vaccine Fr-A β 1-15 and HBc-A β 1-15, respectively. Control sera from non-immunized mice (preimmune sera) were also included. The results for different serum dilutions are shown as optical density at 450 nm. The average of three vaccinated mice is shown. All vaccinated mice contained IgG antibodies specific for A.beta.1-15 in serum.

Example 14

Coupling of Abeta 1-15, Abeta 1-27 and Abeta 33-42 peptides to type I pili

Coupling of A.beta.1-15, A.beta.1-27 and A.beta.33-42 peptides to pili using the cross-linking agent SMPH.

The following a β peptides were chemically synthesized:

DAEFRHDSGYEVHHQGGC ("a β 1-15"), the peptide comprising the amino acid sequence of human a β amino acid residues 1-15, fused at its C-terminus to the sequence GGC for coupling to pili and VLPs;

DAEFRHDSGYEVHHQKLVFFAEDVGSNGGC ("a β 1-27"), the peptide comprising the amino acid sequence of human a β amino acid residues 1-27, fused at its C-terminus to the sequence GGC for coupling to pili and VLPs; and

CGHGNKSGLMVGGVVIA ("a β 33-42"), which peptide comprises the amino acid sequence of human a β amino acid residues 33-42, fused at its N-terminus to the sequence CGHGNKS for coupling to pili and VLPs. All three peptides were chemically coupled to the pili as described below.

2ml of a solution of 2mg/ml pili in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 468. mu.l of a 33.3mM SMPH (Pierce) aqueous solution on a shaker at 25 ℃ for 45 minutes. The reaction solution was applied to a PD10 column (Pharmacia) and eluted with 6X 500. mu.l of 20mM Hepes, 150mM NaCl pH 7.4. The eluted fractions were analyzed by dot hybridization on nitrocellulose (Schleicher & Schuell) and stained with Amidobrack. Fractions 3-6 were combined. The samples were then frozen in equal portions in liquid nitrogen and stored at-80 ℃ until used for coupling.

Mu.l of the thawed desalting reaction mixture was reacted with 200. mu.l of DMSO and 2.5. mu.l each of the corresponding 50mM peptide DMSO stock solutions on a shaker at room temperature for 3.5 hours. 400 ul of the reaction mixture was subsequently dialyzed at 4 ℃ for 1 hour three times against 1L of 20mM Hepes, 150mM NaCl pH7.4 in a dialysis tube with a molecular weight exclusion of 10000 Da. Then, the samples were equally divided and frozen in liquid nitrogen and stored at-80 ℃.

Samples for SDS-PAGE were prepared as follows: 100 μ l of the dialyzed coupling reaction was incubated on ice for 10 minutes in 10% TCA and then centrifuged. The pellet was resuspended in 50. mu.l of 8.5M guanidine hydrochloride solution and incubated at 70 ℃ for 15 minutes. The sample was then precipitated with ethanol and after the second centrifugation step, the pellet was resuspended in sample buffer.

The results of the coupling experiments were analyzed by SDS-PAGE under reducing conditions. The coupling bands for all three peptides were clearly visible, confirming successful coupling of the a β peptide to the pili.

Example 15

Vaccination of APP23 mice with A β peptides coupled to Q β capsid proteins

Immunization of APP23 mice

Three different Ass peptides (Ass 1-27-Gly-Gly-Gly-Cys-NH 2; H-Cys-Gly-His-Gly-Asn-Lys-Ser-Ass 33-42; Ass 1-15-Gly-Gly-Cys-NH2) were coupled to the Q β capsid protein. The resulting vaccines were named "Qb-Ab 1-15", "Qb-Ab 1-27" and "Qb-Ab 33-42". Female APP23 mice carrying the human APP transgene (Sturchler-Pierrat et al, Proc. Natl. Acad. Sci. USA 94: 13287-. Mice were injected subcutaneously with 25 μ g of vaccine diluted in sterile PBS and boosted 14 days later with the same amount of vaccine. Blood was taken from the tail vein of mice before the start of immunization and 7 days after the booster injection. Sera were analyzed by ELISA.

B.ELISA

A.beta.1-40 and A.beta.1-42 peptide stocks were prepared in DMSO and diluted with coating buffer prior to use. ELISA plates were coated with either A.beta.1-40 or A.beta.1-42 peptides at 0.1. mu.g/well. The ELISA plate was blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Sera obtained prior to vaccination were also included as controls. Serum dilutions showing mean values above baseline by 3 standard deviations were calculated and defined as "ELISA titers". All 3 vaccines tested were immunogenic in APP23 mice and induced high antibody titers against a β 1-40 and/or a β 1-42. The results are shown in fig. 20. No specific antibodies were detected in preimmune sera of the same mice (data not shown).

FIG. 20 shows ELISA signals obtained on day 22 using sera from mice immunized with vaccines Fr-A β 1-15 and HBc-A β 1-15, respectively. Control sera from non-immunized mice (preimmune sera) were also included. The results for different serum dilutions are shown as optical density at 450 nm. The average of three vaccinated mice is shown.

Mouse A21-A30 received vaccine Qb-Ab1-15, mouse A31-A40 received Qb-Ab1-27, and mouse A41-49 received Qb-Ab 33-42. Each mouse was assayed for serum antibody titers specific for A β 1-40 and A β 1-42 peptides by ELISA on day 21. Each mouse showed ELISA titers defined as serum dilutions with mean values 3 standard deviations above baseline. The mice vaccinated with Qb-Ab1-15 or Qb-Ab1-27 produced high antibody titers against A β 1-40 and A β 1-42, while the mice vaccinated with Qb-Ab33-42 produced only high antibody titers against A β 1-42 peptide.

Example 16

Coupling of Fab antibody fragments to Q.beta.capsid protein

A solution of 4.0mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with 2.8mM SMPH (Pierce) from stock solution dissolved in DMSO on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times each for 2 hours.

Fab fragments of human IgG generated by papain digestion of human IgG were purchased from Jackson Immunolab. The solution (11.1mg/ml) was diluted to a concentration of 2.5mg/ml with 20mM Hepes, 150mM NaCl pH7.2 and reacted with Dithiothreitol (DTT) or tricarboxyethyl phosphine (TCEP) at different concentrations (0-1000. mu.M) for 30 minutes at 25 ℃.

The derivatized and dialyzed Q.beta.capsid protein solution was mixed with the unreduced or reduced Fab solution (final concentration: 1.14mg/ml Q.beta.and 1.78mg/ml Fab) for induction coupling and reacted overnight on a shaker at 25 ℃.

The reaction products were analyzed on a 16% SDS-PAGE gel under reducing conditions. The gel was stained with Coomassie Brilliant blue. The results are shown in fig. 21.

A coupled product of about 40kDa was detectable in samples where Fab was reduced with 25-1000. mu.M TCEP and 25-100. mu.M DTT prior to coupling (FIG. 21, arrow), whereas reduction with 10. mu.M TCEP, 10. mu.M DTT or 1000. mu.M DTT was not detectable. The coupling band also reacted with anti-Q β antisera (data not shown), clearly confirming the covalent coupling of the Fab fragment to the Q β capsid protein.

The samples loaded onto the gel of fig. 21 were as follows:

lane 1: and (4) molecular weight standard. Lanes 2 and 3: the pre-coupled derivatized Q β capsid protein. Lanes 4-13: q β -Fab coupling reaction after Fab reduction, wherein 4: q beta-Fab coupling reaction after reducing Fab by 10 mu M TCEP; 5: q beta-Fab coupling reaction after reducing Fab by 25 mu M TCEP; 6: q beta-Fab coupling reaction after reducing Fab by 50 mu M TCEP; 7: q beta-Fab coupling reaction after reducing Fab by 100 mu M TCEP; 8: reducing the Fab by 1000 μ M TCEP, and then carrying out Q β -Fab coupling reaction; 9: reducing the Fab by using 10 mu M DTT to perform Q beta-Fab coupling reaction; 10: reducing the Fab by 25 mu M DTT to obtain Q beta-Fab coupling reaction; 11: reducing the Fab by 50 mu M DTT to obtain Q beta-Fab coupling reaction; 12: reducing the Fab by 100 mu M DTT to obtain Q beta-Fab coupling reaction; 13: coupling of Q.beta. -Fab after reduction of Fab with 1000. mu.M DTFT. 14: fab before conjugation. The gel was stained with Coomassie Brilliant blue. Molecular weights of the standard proteins are shown in the left blank. The arrows indicate the coupling bands.

Example 17

Vaccination of APP23 mice with A β peptides coupled to Q β capsid proteins

Immunization of APP23 mice

Three different Ass peptides (Ass 1-27-Gly-Gly-Gly-Cys-NH 2; H-Cys-Gly-His-Gly-Asn-Lys-Ser-Ass 33-42; Ass 1-15-Gly-Gly-Cys-NH2) were coupled to the Q β capsid protein. The resulting vaccines were named "Qb-Ab 1-15", "Qb-Ab 1-27" and "Qb-Ab 33-42". Female APP23 mice carrying the human APP transgene (Sturchler-Pierrat et al, Proc. Natl. Acad. Sci. USA 94: 13287-. Mice were injected subcutaneously with 25 μ g of vaccine diluted in sterile PBS and boosted 14 days later with the same amount of vaccine. Blood was taken from the tail vein of mice before the start of immunization and 7 days after the booster injection. Sera were analyzed by ELISA.

B.ELISA

A.beta.1-40 and A.beta.1-42 peptide stocks were formulated in DMSO and diluted with coating buffer prior to use. ELISA plates were coated with either A.beta.1-40 or A.beta.1-42 peptides at 0.1. mu.g/well. The ELISA plate was blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Sera obtained prior to vaccination were also included as controls. Serum dilutions with mean values 3 standard deviations above baseline were calculated and defined as "ELISA titers". All 3 vaccines tested were immunogenic in APP23 mice and induced high antibody titers against a β 1-40 and/or a β 1-42. The results are shown in fig. 20. No specific antibodies were detected in preimmune sera of the same mice (data not shown).

FIG. 20 shows ELISA signals obtained on day 22 using sera from mice immunized with vaccines Qb-Ab1-15, Qb-Ab1-27, and Qb-Ab33-42, respectively. Mouse A21-A30 received vaccine Qb-Ab1-15, mouse A31-A40 received Qb-Ab1-27, and mouse A41-49 received Qb-Ab 33-42. Each mouse was assayed for serum antibody titers specific for A β 1-40 and A β 1-42 peptides by ELISA on day 21. Each mouse showed ELISA titers defined as serum dilutions with mean values 3 standard deviations above baseline. The mice vaccinated with Qb-Ab1-15 or Qb-Ab1-27 developed high antibody titers against A β 1-40 and A β 1-42, whereas the mice vaccinated with Qb-Ab33-42 only had high antibody titers against A β 1-42 peptide. In a transgenic mouse expressing human Abeta transgene, a very strong immune response is obtained by using human Abeta peptide, and the coupling of the Abeta peptide and Q beta capsid protein is proved to overcome the tolerance to self antigen.

Example 18

Construction, expression and purification of Q beta coat protein mutants

Construction of pQ beta-240

Plasmid pQ β 10(Kozlovska, TM et al, Gene 137: 133-137) was used as the initial plasmid for the construction of pQ β -240. The mutation Lys13 → Arg was generated by inverse PCR. The reverse primer is designed in a reverse tail-tail direction:

5'-GGTAACATCGGTCGAGATGGAAAACAAACTCTGGTCC-3' and

5’-GGACCAGAGTTTGTTTTCCATCTCGACCGATGTTACC-3’。

The product of the first PCR was used as a template for the second PCR reaction using the forward 5'-AGCTCGCCCGGGGATCCTCTAG-3' and the reverse 5'-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3' primers. The product of the second PCR was digested with XbaI and Mph1103I and cloned into pQ β 10 expression vector digested with the same restriction enzymes. The PCR reaction was carried out using PCR kit reagents according to the manufacturer's recommended protocol (MBI Fermentas, Vilnius, Litao-wana).

Sequencing by the directed marker incorporation method confirmed the desired mutation. Coli cells carrying pQ β -240 support efficient synthesis of the 14kD protein, which co-migrates on PAGE with control Q β coat protein isolated from Q β phage particles.

The obtained amino acid sequence is: (SEQ ID NO: 255)

AKLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLI DAIDQLNPAY

Construction of pQ beta-243

Plasmid pQ β 10 was used as the starting plasmid for the construction of pQ β -243. The mutation Asn10 → Lys was generated by inverse PCR. The reverse primer is designed in a reverse tail-tail direction:

5'-GGCAAAATTAGAGACTGTTACTTTAGGTAAGATCGG-3' and

5’-CCGATCTTACCTAAAGTAACAGTCTCTAATTTTGCC-3’。

the product of the first PCR was used as a template for the second PCR reaction using the forward 5'-AGCTCGCCCGGGGATCCTCTAG-3' and the reverse 5'-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3' primers. The product of the second PCR was digested with XbaI and Mph1103I and cloned into pQ β 10 expression vector digested with the same restriction enzymes. The PCR reaction was carried out using PCR kit reagents according to the manufacturer's recommended protocol (MBI Fermentas, Vilnius, Litao-wana).

Sequencing by the directed marker incorporation method confirmed the desired mutation. Coli cells carrying pQ β -243 support efficient synthesis of the 14kD protein, which co-migrates on PAGE with control Q β coat protein isolated from Q β phage particles.

The obtained amino acid sequence is: (SEQ ID NO: 256)

AKLETVTLGKIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQ beta-250

Plasmid pQ β -240 was used as the starting plasmid for the construction of pQ β -250. The mutation Lys2 → Arg was generated by site-directed mutagenesis. Synthesis of mutant PCR fragments Using upstream primer 5'-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3' and downstream primer 5'-GATTTAGGTGACACTATAG-3', introduced into pQ β -185 expression vector at the only restriction sites NcoI and HindIII. The PCR reaction was carried out using PCR kit reagents according to the manufacturer's recommended protocol (MBI Fermentas, Vilnius, Litao-wana).

Sequencing by the directed marker incorporation method confirmed the desired mutation. Coli cells carrying pQ β -250 support efficient synthesis of the 14kD protein, which co-migrates on PAGE with control Q β coat protein isolated from Q β phage particles.

The obtained amino acid sequence is: (SEQ ID NO: 257)

ARLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQ beta-251

Plasmid pQ β 10 was used as the starting plasmid for the construction of pQ β -251. The mutation Lys16 → Arg was generated by inverse PCR. The reverse primer is designed in a reverse tail-tail direction:

5'-GATGGACGTCAAACTCTGGTCCTCAATCCGCGTGGGG-3' and

5’-CCCCACGCGGATTGAGGACCAGAGTTTGACGTCCATC-3’。

the product of the first PCR was used as a template for the second PCR reaction using the forward 5'-AGCTCGCCCGGGGATCCTCTAG-3' and the reverse 5'-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3' primers. The product of the second PCR was digested with XbaI and Mph1103I and cloned into pQ β 10 expression vector digested with the same restriction enzymes. The PCR reaction was carried out using PCR kit reagents according to the manufacturer's recommended protocol (MBI Fermentas, Vilnius, Litao-wana).

Sequencing by the directed marker incorporation method confirmed the desired mutation. Coli cells carrying pQ β -251 support efficient synthesis of the 14kD protein, which co-migrates on PAGE with control Q β coat protein isolated from Q β phage particles. The amino acid sequence encoded by the construct was obtained as set forth in SEQ ID NO: 259 is shown.

Construction of pQ β -259

Plasmid pQ β -251 was used as the starting plasmid for the construction of pQ β -259. The mutation Lys2 → Arg was generated by site-directed mutagenesis. Synthesis of mutant PCR fragments Using upstream primer 5'-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3' and downstream primer 5'-GATTTAGGTGACACTATAG-3', introduced into pQ β -185 expression vector at the only restriction sites NcoI and HindIII. The PCR reaction was carried out using a PCR kit according to the manufacturer's recommended protocol (MBIFermestas, Vilnius, Alexania).

Sequencing by the directed marker incorporation method confirmed the desired mutation. Coli cells carrying pQ β -259 support efficient synthesis of the 14kD protein, which co-migrates on PAGE with control Q β coat protein isolated from Q β phage particles.

The obtained amino acid sequence is: (SEQ ID NO: 258)

AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

General methods for expression and purification of Q β and Q β mutants

Escherichia coli JM109 was transformed with Q.beta.expression plasmid. The clone transformed with Q.beta.expression plasmid was inoculated in 5ml of LB liquid medium containing 20. multidot.g/ml ampicillin. Incubate at 37 ℃ for 16-24 hours without shaking.

The prepared bacterial culture was inoculated at 1: 100 into 100-300ml of LB medium containing 20. g/ml ampicillin. Incubate overnight at 37 ℃ without shaking. The prepared bacterial culture was inoculated to M9+ 1% casamino acid + 0.2% glucose medium in a flask at a ratio of 1: 50, and incubated overnight at 37 ℃ with shaking.

Purification of

Solutions and buffers for purification processes

1. Lysis buffer LB

50mM Tris-HCl pH8.0, containing 5mM EDTA, 0.1% triton X100 and freshly prepared PMSF, to 5. mu.g/ml. Does not contain lysozyme and DNAse.

2.SAS

Saturated aqueous ammonium sulfate solution

3. Buffer NET

20mM Tris-HCl pH7.8, containing 5mM EDTA and 150mM NaCl.

4.PEG

40% (w/v) polyethylene glycol 6000 dissolved in NET

Crushing and cracking

The frozen cells were resuspended in LB at 2ml/g cells. The mixture was sonicated at 22kH 5 times for 15 seconds each, with 1 minute intervals, and the solution was cooled on ice. The lysate was then centrifuged for 1 hour at 14000rpm using a Jaecki K60 rotor. Unless otherwise indicated, the centrifugation steps described below were all performed with the same rotor. The supernatant was stored at 4 ℃ and the cell debris was washed twice with LB. After centrifugation, the supernatant and wash of the lysate were combined.

Fractionation

To the above combined lysates was added dropwise saturated ammonium sulfate solution under stirring. The volume of the SAS was adjusted to 1/5 of the total volume to obtain 20% saturation. The solution was left to stand overnight and centrifuged the next day at 14000rpm for 20 minutes. The precipitate was washed with a small amount of 20% ammonium sulfate and centrifuged again. The obtained supernatants were combined and SAS was added dropwise to obtain 40% saturation. The solution was left to stand overnight and centrifuged the next day at 14000rpm for 20 minutes. The obtained precipitate was dissolved with NET buffer.

Chromatography

Capsid proteins re-dissolved in NET buffer were loaded onto a Sepharose CL-4B column. 3 peaks eluted during chromatography. The first contained mainly membranes and membrane fragments, not collected. The capsid is contained in the second peak, while the third peak contains other E.coli proteins.

The peak fractions were pooled and the NaCl concentration was adjusted to a final concentration of 0.65M. The PEG solution in a volume corresponding to half of the combined peak fractions was added dropwise with stirring. The solution was left overnight without stirring. The capsid protein is precipitated by centrifugation at 14000rpm for 20 minutes. Then dissolved in the minimum volume of NET and loaded again onto a Sepharose CL-4B column. The peak fractions were combined and precipitated with 60% saturated (w/v) ammonium sulfate. After centrifugation and resolubilization in NET buffer, the capsid proteins were loaded onto a Sepharose CL-6B column for renewed chromatography.

Dialyzing and drying

The peak fractions obtained as above were combined, dialyzed thoroughly against sterile water, lyophilized for storage.

Expression and purification of Q beta-240

Cells (E.coli JM109 transformed with plasmid pQ. beta. -240) were resuspended in LB, sonicated 5 times for 15 seconds each (water ice jacket), and centrifuged at 13000rpm for 1 hour. The supernatant was stored at 4 ℃ until further processing, and the splits were washed 2 times with 9ml LB and finally with 9ml 0.7M urea dissolved in LB. All supernatants were pooled and applied to a Sepharose CL-4B column. The combined peak fractions were precipitated with ammonium sulfate and centrifuged. The resolubilized protein was then further purified on a Sepharose 2B column and finally on a Sepharose 6B column. The capsid peaks were finally extensively dialyzed against water and lyophilized as described above. The assembly of coat proteins into capsids was confirmed by electron microscopy.

Expression and purification of Q beta-243

Cells (E.coli RR1) were resuspended in LB and processed as described in general methods. The protein was purified by successive gel filtration steps using a Sepharose CL-4B column and finally a Sepharose CL-2B column. The peak fractions were pooled and lyophilized as described above. The assembly of coat proteins into capsids was confirmed by electron microscopy.

Expression and purification of Q beta-250

Cells (pQ. beta. -250 transformed E.coli JM109) were resuspended in LB and treated as described above. The protein was purified by gel filtration on a Sepharose CL-4B column and finally on a Sepharose CL-2B column and lyophilized as described above. The assembly of coat proteins into capsids was confirmed by electron microscopy.

Expression and purification of Q beta-259

Cells (pQ. beta. -259 transformed E.coli JM109) were resuspended in LB and sonicated. The splits were washed once with 10ml LB and a second time with 10ml 0.7M urea dissolved in LB. The protein was purified by two gel filtration chromatography steps using Sepharose CL-4B columns.

Proteins were dialyzed and lyophilized as described above. The assembly of coat proteins into capsids was confirmed by electron microscopy.

Example 19

Desensitization of allergic mice with PLA2 coupled to Q β capsid protein

A. Desensitizing allergic mice by vaccination

Female CBA/J mice (8 weeks old) were sensitized with PLA 2: each mouse was adsorbed 0.1. mu.g of PLA2(Latoxan, France) onto 1mg of alum (Imject, Pierce) in a total volume of 66. mu.l by shaking for 30 minutes, followed by subcutaneous injection. This step was repeated every 14 days for a total of 4 times. This treatment resulted in the production of PLA 2-specific serum IgE, but did not produce IgG2a antibodies. 1 month after the last sensitization, mice were injected subcutaneously with 10 μ g of vaccine containing recombinant PLA2 coupled to Q β capsid protein. After 1 and 2 weeks, the vaccine was again treated with the same amount of vaccine. After 1 week of the last treatment, blood was collected from the mice and then challenged intraperitoneally with 25 μ g PLA2(Latoxan) and rectal temperature was measured with a calibrated digital thermometer. Sensitized mice that were not treated with Q β capsid protein-PLA 2 served as controls. While all control mice experienced an allergic reaction, as evidenced by a significant decrease in rectal temperature following PLA2 challenge, vaccinated mice were fully or at least partially protected. The results are shown in fig. 25A.

B.ELISA

ELISA plates (Maxisorp, Nunc) were coated with 5. mu.g/ml PLA2 (Latoxan). The plates were blocked and then incubated with serial dilutions of serum. For the detection of IgE antibodies, the sera were pretreated with protein G beads (Pharmacia) at room temperature on a shaker for 60 minutes. The beads were removed by centrifugation and the supernatant used for ELISA. Antibodies that bind to PLA2 were detected with enzyme-labeled anti-mouse IgG2a or IgE antibodies. The ELISA titers were determined at half maximal optical density (OD 50%), representing-log 5 for IgG2a and-log 5 for IgE for 100-fold pre-diluted serum. For all mice, IgG2a and IgE specific for PLA2 in serum were determined before and at the end of the vaccine treatment. Vaccination resulted in a significant increase in PLA 2-specific IgG2a, while no change in IgE titers was noted. These results indicate that vaccination results in induction of a Th 1-like immune response (reflected by IgG2a production). The results are shown in fig. 25B.

The allergic reactions of vaccinated and non-vaccinated mice are shown in figure 25A.

Mice were sensitized with PLA2 and then treated 3 times subcutaneously with 10 μ g of a vaccine containing PLA2 coupled to Q β capsid protein. Control mice were sensitized but not vaccinated. 1 week after the last inoculation, all mice were challenged intraperitoneally with 25 μ g PLA2 and the allergic reaction was monitored by measuring rectal temperature for 60 minutes. Although all control mice showed a significant reduction in body temperature, the vaccinated mice were completely or at least partially protected from allergic reactions.

Induction of PLA 2-specific IgG2a by vaccination is shown in fig. 25B.

Mice were sensitized with PLA2 and then treated 3 times with 10 μ g of a vaccine containing PLA2 coupled to Q β capsid protein. Control mice were sensitized but not vaccinated. Serum from sensitized mice was collected before the start of treatment, after the end of treatment, and before challenge. In vaccinated mice (left panel), a significant increase in PLA 2-specific IgG2a was observed.

Example 20

PLA₂Cys (also called PLA)₂Fusion protein), refolding, purification, and conjugation

A. Expression and preparation of Inclusion bodies

With PLA containing example xxx₂pET11a plasmid for the Cys gene transformed E.coli BL21 DE3Rill (Stratagene). The overnight cultures were grown in dYT medium containing 100. mu.g/ml ampicillin and 15. mu.g/ml chloramphenicol. The culture solution is diluted by fresh dYT culture medium containing ampicillin and chloramphenicol Growth at 37 ℃ until OD_600mn1. The culture broth was induced with 1mM IPTG and cultured for an additional 4 hours. The cells were harvested by centrifugation and resuspended in PBS buffer containing 0.5mg/ml lysozyme. After incubation on ice, cells were sonicated on ice and MgCl was added₂To a concentration of 10 mM. To the cell lysate, 6. mu.l of Benzonase (Merck) was added and the lysate was incubated at room temperature for 30 minutes. Triton was added to a final concentration of 1% and the lysates were incubated on ice for an additional 30 min. The inclusion body (1B) pellet was collected by centrifugation at 13000g for 10 minutes. The inclusion body pellet was washed with a washing buffer containing 20mM Tris, 23% sucrose, 1mM EDTA, pH 8.0. Inclusion bodies were solubilized in 6M guanidinium hydrochloride, 20mM Tris, pH8.0, containing 200mM DTT. Solubilized inclusion bodies were centrifuged at 50000g and the supernatant was dialyzed against 6M guanidine hydrochloride, 20mM Tris, pH8.0, followed by dialysis against the same buffer containing 0.1mM DTT. Oxidized glutathione was added to a final concentration of 50mM and the solubilized inclusion bodies were incubated at room temperature for 1 hour. Solubilized inclusion bodies were dialyzed against 6M guanidine hydrochloride, 20mM Tris, pH 8.0. The concentration of the inclusion body solution was estimated by Bradford analysis and SDS-PAGE.

B. Refolding and purification

The inclusion body solution was added slowly in three portions every 24 hours to a refolding buffer containing 2mM EDTA, 0.2mM benzamidine, 0.2mM6 aminocaproic acid, 0.2mM guanidine hydrochloride, 0.4 ML-arginine, pH6.8 to a final concentration of 3. mu.M, to which 5mM reduced glutathione and 0.5mM oxidized glutathione were added at 4 ℃ before refolding started. The refolded solution was concentrated to half its volume by ultrafiltration using YM10 membrane (Millipore) and dialyzed against PBs containing 0.1mM DTT, pH 7.2. The protein was further concentrated by ultrafiltration and purified by loading onto a Superdex G-75 column (Pharmacia) equilibrated with 20mM Hepes, 150mM NaCl, 0.1mM DTT at 4 ℃. The pH of the equilibration buffer was adjusted to 7.2 at room temperature. Fractions of single fractions (monomeric fractions) were pooled.

C. Coupling of

A solution of 1.5mg Q.beta.in 0.75mL 20mM Hepes, 150mM NaCl, pH7.4 was reacted with 0.06mL Sulfo-SMPB (Pierce; 31mM water stock) for 45 minutes at room temperature. The reaction mixture was dialyzed overnight against 20mM Hepes, 150mM NaCl, pH7.4, and 0.75mL of this solution was mixed with 1.5mL of a solution of PLA2-Cys in 0.1mM DTT (62. mu.M) and 0.43mL of 20mM Hepes, 150mM NaCl, 137. mu.M DTT adjusted to pH7.4 at room temperature. The coupling reaction was continued at room temperature for 4 hours and dialyzed overnight against 20mM Hepes, 150mM NaCl, pH7.4 using a Spectra Por dialysis tube (Spectrum) with a molecular weight cut-off of 300000 Da. The coupling reaction was analyzed by SDS-PAGE Coomassie blue staining and Western blot using rabbit anti-bee venom antiserum (diluted 1: 10000) and developed with goat anti-rabbit alkaline phosphatase conjugate (diluted 1: 10000); alternatively, rabbit anti-Q.beta.antiserum (1: 5000) was used and goat anti-rabbit alkaline phosphatase conjugate (1: 10000 dilution) was used for color development. In both cases, the sample was reacted under reducing conditions.

The results of the coupling reaction are shown in fig. 26. Corresponding to Q beta capsid protein and PLA₂The band of-Cys coupled product is between Q beta capsid protein and PLA ₂Coomassie blue staining of the inter-Cys coupling reactions, clearly visible on SDS-PAGE (left panel), anti-Q β Western blot (middle panel) and anti-PLA 2 Western blot (right panel), indicated by arrows. Mu.l of the coupling reaction solution and 50. mu.l of the dialyzed coupling reaction solution were applied to the gel.

Lane 1: and (4) protein standard. 2: dialyzed coupling reaction solution 1. 3: coupling reaction 1. 4: coupling reaction 2. 5: coupling reaction 2. 6: coupling reaction 1. 7: dialyzed coupling reaction solution 1. 8: and (4) protein standard. 9: coupling reaction 2. 10: coupling reaction 1. 11: dialyzed coupling reaction solution 1. 12: and (4) protein standard.

Example 21

Coupling of anti-idiotype IgE mimobody VAE051 to Q beta,

Immunization of mice and detection of antisera

A solution of 4.0mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 10-fold molar excess of SMPH (Pierce) (from a 100mM stock solution dissolved in DMSO) on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The VAE051 solution (2.4mg/ml) was reduced with equimolar concentrations of TCEP at 25 ℃ for 60 minutes.

Mu.l of the dialyzed Q.beta.reaction mixture was reacted with 340. mu.l of TCEP-treated VAE051 solution (2.4mg/ml) in a total volume of 680. mu.l of 50mM sodium acetate buffer on a shaker at 16 ℃ for 2 hours.

The reaction products were analyzed on a 16% SDS-PAGE gel under reducing conditions. The gel was stained with Coomassie Brilliant blue. The other two bands (not in VAE or Q β solution) in the coupling reaction represent the heavy and light chains of VAE051 coupled to Q β (fig. 28A). The identity of these two bands was confirmed by Western blotting with heavy and light chain specific antibodies, respectively.

Immunization of mice

Using a membrane with a cutoff of 300000Da, the Q β -VAE051 coupled solution was dialyzed against 20mM hepes, 150mM NaCl, pH 7.2. Two female Balb/c mice were injected intraperitoneally with 50 μ g Q β -VAE051 on days 0 and 14. Mice were bled retro-orbitally on day 28 and their sera were analyzed by IgE-and VAE 051-specific ELISA.

ELISA

ELISA plates were coated with human IgE at a concentration of 0.8. mu.g/ml or VAE051 at 10. mu.g/ml. The ELISA plate was blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody (fig. 28B).

Both mice showed strong reactivity to VAE051 as well as human IgE. Preimmune sera of the same mice did not show any reactivity to VAE051 and IgE (fig. 28B). This confirms the production of antibodies against the anti-idiotype IgE mimobody VAE051, which also recognize the "parent" molecule IgE.

Example 22

High occupancy coupling of DerpI peptide to wild-type Q beta capsid protein using cross-linker SMPH

The Derp1, 2 peptide was chemically synthesized, and for coupling purposes, a cysteine was added to the N-terminus, having the following sequence: H2N-CQIYPPNANKIREA LAQTHSA-COOH. This peptide was used to chemically couple to wild-type Q β capsid protein as described below.

Coupling of flag peptide to Q beta capsid protein

A solution of Q.beta.capsids at a concentration of 2mg/ml in 20mM Hepes, 150mM NaCl pH7.2 was reacted with a 5-fold or 20-fold excess of the cross-linking agent SMPH (Pierce) for 30 minutes at 25 ℃ on a shaker. The reaction solution was subsequently dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. The dialyzed reaction mixture was reacted with 5-fold excess of Derp1, 2 peptide on a shaker at 25 ℃ for 2 hours.

The results of the coupling reaction are shown in fig. 24. The coupling bands corresponding to 1, 2, 3 peptides per subunit, respectively, are clearly visible on the gel, as indicated by the arrows. On average two peptides per subunit are displayed on the capsid.

The samples loaded onto the gel of fig. 24 were as follows:

lane 1: and (4) protein standard. 2: q β capsid protein derived with a 5-fold excess of SMPH. 3: SMPH-derived Q β capsid protein was used in a 20-fold excess. 4: coupling reaction of 5-fold derivatized Q β capsid protein. 5: coupling reaction of 20-fold derivatized Q β capsid protein.

Example 23

Insertion of lysine residue-containing peptide into HBcAg (1-149) c/e1 epitope

The c/e1 epitope (residues 72-88) of HBcAg is located in the top region of the hepatitis B virus capsid (HBcAg) surface. Part of this region (proline 79 and alanine 80) was genetically replaced with the peptide Gly-Lys-Gly (HBcAg-Lys construct). The introduced lysine residue contains a reactive amino group on its side chain, which can be used for intermolecular chemical crosslinking of HBcAg particles with any antigen containing a free cysteine group.

Has the sequence shown in SEQ ID NO: 158 was generated by PCR using HBcAg-LysDNA having the amino acid sequence shown in seq id no: two fragments encoding the HBcAg fragment (amino acid residues 1-78 and 81-149) were amplified separately by PCR. The primers used in these PCRs also incorporate DNA sequences encoding Gly-Gly-Lys-Gly-Gly peptides. The HBcAg (1-78) fragment was amplified from pEco63 using the primers EcoRiHBcAg(s) and Lys-HBcAg (as). The HBcAg (81-149) fragment was amplified from pEco63 using primers Lys-HBcAg(s) and HBcAg (1-149) hind (as). Primers Lys-HBcAg (as) and Lys-HBcAg(s) introduce complementary DNA sequences at the ends of the two PCR products, allowing the two PCR products to fuse in a subsequent assembly PCR. The assembled fragment was amplified by PCR using the primers EcoRI HBcAg(s) and HBcAg (1-149) hind (as).

For the PCR reaction, 2 units of Pwo polymerase, 0.1mM dNTPs and 2mM MgSO were added₄100pmol each of the oligonucleotides and 50ng of the template DNA were used in 50. mu.l of the reaction mixture. The temperature cycles of the two reactions were carried out as follows: 2 minutes at 94 ℃; 94 deg.C (1 min), 50 deg.C (1 min), 72 deg.C (2 min) for 30 cycles.

The primer sequence is as follows:

EcoRIHBcAg(s)：

(5’-CCGGAATTCATGGACATTGACCCTTATAAAG-3’)(SEQ ID NO：79)；

Lys-HBcAg(Es)：

(5’-

CCTAGAGCCACCTTTGCCACCATCTTCTAAATTAGTACCCACCCAGGTAGC-3’)(SEQ ID NO：80)；

Lys-HBcAg(s)：

(5’-

GAAGATGGTGGCAAAGGTGGCTCTAGGGACCTAGTAGTCAGTTATGTC-3’)(SEQ ID NO：81)；

HbcAg(1-149)Hind(as)：

(5’-CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAG-3’)(SEQID NO：82).

to fuse two PCR fragments by PCR, the fusion product was mixed with 2 units of Pwo polymerase at 0.1mM dNTPs and 2mM MgSO₄100pmol each of the primers EcoRiHBcAg(s) and HBcAg (1-149) hind (as) was used in 50. mu.l of the reaction mixture, and 100ng of the two purified PCR fragments were contained. The PCR cycling conditions were: 2 minutes at 94 ℃; 94 deg.C (1 min), 50 deg.C (1 min), 72 deg.C (2 min) for 30 cycles. The assembled PCR products were analyzed by agarose gel electrophoresis, purified, and digested for 19 hours in the appropriate buffer containing EcoRI and HindIII restriction enzymes. The digested DNA fragment was ligated into the EcoRI-HindIII digested pKK vector, resulting in pKK-HBcAg-Lys expression vector. The insertion of the PCR product into the vector was analyzed by EcoRI/HndIII restriction and DNA sequencing of the insert.

Example 24

Expression and partial purification of HBcAg-Lys

Coli XL-1blue strain was transformed with pKK-HBcAg-Lys. 1ml of the overnight culture of the bacteria was inoculated into 100ml of LB medium containing 100. mu.g/ml ampicillin. The culture was incubated at 37 ℃ for 4 hours until an OD of about 0.8 at 600nm was reached. Synthesis of HBcAg-Lys was induced by adding IPTG to a final concentration of 1 mM. After induction, the bacteria were shaken for a further 16 hours at 37 ℃. The bacteria were collected by centrifugation at 5000 Xg for 15 minutes. The precipitate was frozen at-20 ℃. The pellet was thawed and resuspended in bacterial lysis buffer (10mM Na) supplemented with 200. mu.g/ml lysozyme and 10. mu.l Benzonase (Merck) ₂HPO₄pH7.0, 30mM NaCl, 0.25% Tween-20, 10mM EDTA, 10mM DTT). Cells were incubated at room temperature for 30 minutes and disrupted with a French press. Triton X-100 was added to the lysate to a final concentration of 0.2% lysate and incubated on ice for 30 minutes with occasional shaking. IPTG induces expression of HBcAg-Lys in E.coli cells carrying pKK-HBcAg-Lys expression plasmid or a control plasmid. Samples were taken from the culture of the bacteria carrying the pKK-HBcAg-Lys plasmid and the control plasmid before IPTG addition. After 16 hours of IPTG addition, samples were taken from the culture medium containing pKK-HBcAg-Lys and the control culture medium. Protein expression was monitored by SDS-PAGE Coomassie blue staining.

The lysate was then centrifuged at 12000 Xg for 30 min in order to remove insoluble cell debris. The supernatant and pellet were analyzed by Western blotting using a monoclonal antibody against HBcAg (YVS1841, available from Accurate Chemical and scientific Corp., Westbury, NY, USA) and showed that a significant amount of HBcAg-Lys protein was soluble. Briefly, lysates of HBcAg-Lys expressing E.coli cells and control cells were centrifuged at 14000 Xg for 30 min. The supernatant (═ soluble fraction) and pellet (═ insoluble fraction) were separated and diluted in equal volumes with SDS sample buffer. Samples were analyzed by SDS-PAGE followed by Western blotting using anti-HBcAg monoclonal antibody YVS 1841.

The clarified cell lysate was subjected to step gradient centrifugation using a sucrose step gradient consisting of 4ml of 65% sucrose solution overlaid with 3ml of 15% sucrose solution, followed by the addition of 4ml of bacterial lysate. The samples were centrifuged at 100000 Xg for 3 hours at 4 ℃. After centrifugation, 1ml fractions were collected from the top of the gradient and analyzed by SDS-PAGE Coomassie blue staining. HBcAg-Lys protein was detected by Coomassie blue staining.

The HBcAg-Lys protein was enriched at the interface between 15% and 65% sucrose, indicating that capsid particles had formed. Most of the bacterial protein remained in the upper layer of the sucrose-free gradient, so fractional gradient centrifugation of HBcAg-Lys particles resulted in enrichment and partial purification of the particles.

Example 25

Chemical coupling of the FLAG peptide to HBcAg-Lys using the heterobifunctional crosslinker SPDP

To elicit an immune response against the FLAG peptide, a synthetic FLAG peptide containing a cysteine residue at the amino terminus (amino acid sequence CGGDYKDDDDK (SEQ ID NO: 147)) was chemically coupled to purified HBcAg-Lys particles. 600ml of 95% pure HBcAg-Lys particle solution (2mg/ml) were incubated with the heterobifunctional crosslinker N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) (0.5mM) for 30 min at room temperature. After completion of the reaction, the mixture was dialyzed overnight against 1 liter of 50mM phosphate buffer (pH7.2) containing 150mM NaCl to remove free SPDP. To prevent metal-catalyzed thiol oxidation, 500ml of derivatized HBcAg-Lys capsid (2mg/ml) was mixed with 0.1mM FLAG peptide (containing an amino-terminal cysteine) in the presence of 10mM EDTA. Since SPDP releases pyridine-2-thione upon reaction with the free cysteine of the peptide, the reaction was monitored by the increase in optical density at 343nm of the solution. The reaction of the derivatized Lys residue with the peptide is complete after about 30 minutes.

Mice were injected with FLAG-modified particles.

Example 26

Construction of pMPSV-gp140cys

The gp140 gene was amplified by PCR from pCytTSgp140FOS using the oligonucleotides gp140CysEcoRI and SalIgp 140. For PCR, the PCR reaction was carried out in a medium containing 2 units of Pwo polymerase, 0.1mM dNTPs and 2mM MgSO₄100pmol each of the oligonucleotides and 50ng of the template DNA were used in 50ml of the reaction mixture. The temperature cycles of the two reactions were carried out as follows: 2 minutes at 94 ℃; 94 deg.C (0.5 min), 55 deg.C (0.5 min), 72 deg.C (2 min) for 30 cycles.

The PCR product was purified using the QiaEXII kit, digested with SalI/EcoRI and ligated into the vector pMPSVHE digested with the same enzymes.

The oligonucleotide sequence:

gp140CysEcoRI：

5’-GCCGAATTCCTAGCAGCTAGCACCGAATTTATCTAA-3’(SEQ ID NO：83)

SalIgp140：

5’-GGTTAAGTCGACATGAGAGTGAAGGAGAAATAT-3’(SEQ ID NO：84)。

example 27

Expression of pMPSVgp140cys

pMPSVgp140cys (20. mu.g) was linearized by restriction enzyme digestion. The reaction was stopped by phenol/chloroform extraction followed by isopropanol precipitation of the linearized DNA. Restriction enzyme digestion was assessed by agarose gel electrophoresis. For transfection, 5.4. mu.g of linearized pMPSVgp140cys was combined with 0.6. mu.g of linearized pSV2Neo at 30. mu. l H₂In OMix and then add 30. mu.l 1M CaCl₂And (3) solution. Mu.l of phosphate buffer (50mM HEPES, 280mM NaCl, 1.5mM Na) was added₂HPO₄Ph7.05), the solution was shaken for 5 seconds and then incubated at room temperature for 25 seconds. The solution was immediately added to 2ml of HP-1 medium (2% FCS medium) containing 2% FCS. The medium (6-well plate) of BHK21 cells grown to 80% confluence was then replaced with DNA-containing medium. In CO ₂After incubation at 37 ℃ for 5 hours in an incubator, the medium containing DNA was removed and replaced with 2ml of 2% FCS medium containing 15% glycerol. After 30 seconds of incubation the glycerol-containing medium was removed and the cells were rinsed with 5ml of HP-1 medium containing 10% FCS. Finally 2ml of fresh HP-1 medium containing 10% FCS was added.

Selection of stably transfected cells at 37 ℃ CO₂Growth in selection medium (HP-1 medium with G418 added) was carried out in an incubator. When the mixed population was grown to confluence, the culture broth was split between two plates and then grown at 37 ℃ for 12 hours. One cell plate was transferred to 30 ℃ to induce the expression of soluble GP 140-FOS. The other plate was kept at 37 ℃.

Expression of soluble GP140-Cys was determined by Western blot analysis. The medium (0.5ml) was pelleted with methanol/chloroform and the pellet resuspended in SDS-PAGE loading buffer. The sample was heated at 95 ℃ for 5 minutes before loading on a 15% acrylamide gel. For example, "protein function: utility methods described in IRL Press, Oxford (1997), PAGEs 29-55, on SDS-PAGE, proteins were transferred to Protan nitrocellulose membranes (Schleicher & Schuell, Germany). Membranes were blocked with 1% bovine albumin (Sigma) dissolved in TBS (10 XTSB: 87.7g NaCl, 66.1g Trizma hydrochloride (Sigma) and 9.7g Trizma base (Sigma), pH7.4 per liter) for 1 hour at room temperature and then incubated with anti-GP 140 or GP-160 antibodies for 1 hour. The blot was washed 3 times with TBS-T (TBS containing 0.05% Tween 20) for 10 min each and incubated with alkaline phosphatase-anti-mouse/rabbit/monkey/human IgG conjugate for 1 h. After washing 2 times 10 minutes with TBS-T and 2 times 10 minutes with TBS, a color reaction was carried out with an alkaline phosphatase detection reagent (10ml of AP buffer (100mM Tris/HCl, 100mM NaCl, pH9.5)) and 50. mu.l of NBT solution (7.7% nitroblue tetrazolium (Sigma) in 70% dimethylformamide) and 37. mu. l X-phosphate solution (5% 5-bromo-4-chloro-3-indolphosphate in dimethylformamide).

Example 28

Purification of gp140Cys

The anti-gp 120 antibody was covalently coupled to NHS/EDC activated dextran and the column was packed. The supernatant containing GP140Cys was loaded onto the column and after extensive washing, GP140Cys was eluted with 0.1M HCl. During the collection procedure the eluate was directly neutralized with 1M Tris pH7.2 in the collection tube.

Disulfide bonds may form during purification, so collected samples were treated with 10mM DTT in 10mM Tris pH7.5 for 2 hours at 25 ℃.

Followed by 10mM Mes; DTT was removed by dialysis against 80mM NaCl pH 6.0. Finally, as described in example 16, GP140Cys was mixed with alphavirus particles containing JUN residues in E2.

Example 29

PLA₂Construction of-Cys

Amplification of PLA from pAV3PLAfos by PCR Using the oligonucleotides EcoRIPLA and PLA-Cys-hind₂A gene. For PCR, the PCR reaction was carried out in a medium containing 2 units of Pwo polymerase, 0.1mM dNTPs and 2mM MgSO₄100pmol each of the oligonucleotides and 50ng of the template DNA were used in 50. mu.l of the reaction mixture. The temperature cycle of the reaction was carried out as follows: 2 minutes at 94 ℃; 94 deg.C (0.5 min), 55 deg.C (0.5 min), 72 deg.C (2 min) for 30 cycles.

The PCR product was purified using the QiaEXII kit, digested with EcoRI/HindIII, and ligated into the same enzymatically digested vector pAV 3.

Oligonucleotides

EcoRIPLA：

5’-TAACCGAATTCAGGAGGTAAAAAGATATGG-3’(SEQ ID NO：85)

PLA-Cys-hind：

5’-GAAGTAAAGCTTTTAACCACCGCAACCACCAGAAG’3’(SEQ ID NO：86)。

Example 30

PLA₂-Cys expression and purification

In order to produce proteins containing a Cys tail in the cytoplasm, E.coli XL-1Blue strain was transformed with the vectors pAV3:: PLA and pPLA-Cys. The culture broth was incubated at 37 ℃ with shaking in an enrichment medium containing ampicillin. At optical density (550nm), 1mM IPTG was added and incubation was continued for 5 hours. The cells were collected by centrifugation, resuspended in an appropriate buffer (e.g., Tris-HCl, pH7.2, 150mM NaCl) containing DNAse, RNAse and lysozyme, and disrupted by a French press. After centrifugation (Sorvall RC-5C, SS34 rotor, 1500rpm, 10min, 4 ℃), the pellet was resuspended in 25ml inclusion body wash buffer (20mM tris-HCl, 23% sucrose, 0.5% Triton X-100, 1mM EDTA, pH8) at 4 ℃ and centrifuged again as described above. This step was repeated until the supernatant was substantially clear after centrifugation. The inclusion bodies were resuspended in 20ml of lysis buffer (5.5M guanidine hydrochloride, 25mM tris-HCl, pH7.5) at room temperature, the insoluble material was removed by centrifugation, and the supernatant was passed through a sterile filter (0.45 μ M). The protein solution was kept standing at 4 ℃ for at least 10 hours in the presence of 10mM EDTA and 100mM DTT, and then dialyzed 3 times against 10 volumes of 5.5M guanidine hydrochloride, 25mM tris-HCl, 10mM EDTA, pH 6. The solution was dialyzed 2 times against 5L 2M urea, 4mM EDTA, 0.1M NH4Cl, 20mM sodium borate (pH8.3) containing the appropriate redox shuttle (oxidized glutathione/reduced glutathione; cystine/cysteine). Ion exchange chromatography of the refolded protein. The protein is stored in a suitable buffer containing 2-10mM DTT at a pH above 7 to maintain the cysteine residues in a reduced state. The protein solution was passed through a Sephadex G-25 gel filtration column to remove DTT before the protein was coupled to alphavirus particles.

Example 31

Construction of HBcAg containing one inserted lysine residue without free cysteine residue

In a nucleic acid sequence corresponding to SEQ ID NO: 134, which does not contain cysteine residues at positions 48 and 107 and contains an inserted lysine residue (HBcAg), herein referred to as HBcAg-lys-2cys-Mut, was constructed in the following manner.

Three fragments of the HBcAg-Lys gene prepared as described in example 23 were first amplified separately using the following PCR primer combinations, introducing two mutations. The PCR method was carried out essentially as described in example 1, using the conventional cloning technique to prepare the HBcAg-lys-2cys-Mut gene.

Briefly, fragment 1 was prepared with the following primers:

primer 1: EcoRiHBcAg(s)

CCGGAATTCATGGACATTGACCCTTATAAAG(SEQ ID NO：148)

Primer 2: 48as

GTGCAGTATGGTGAGGTGAGGAATGCTCAGGAGACTC(SEQ ID NO：149)

Fragment 2 was prepared with the following primers:

primer 3: 48s

GSGTCTCCTGAGCATTCCTCACCTCACCATACTGCAC(SEQ ID NO：150)

Primer 4: 107as

CTTCCAAAAGTGAGGGAAGAAATGTGAAACCAC(SEQ ID NO：151)

Fragment 3 was prepared with the following primers:

primer 5: HBcAg149 hit-as

CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAGCGTTGATAG(SEQ ID NO：152)

Primer 6: 107s

GTGGTTTCACATTTCTTCCCTCACTTTTGGAAG(SEQ ID NO：153)。

Fragments 1 and 2 were then combined by PCR using the primers EcoRiHBcAg(s) and 107as to generate fragment 4. Fragment 4 and fragment 3 were combined by PCR using the primers EcoRiHBcAg(s) and EcoRiHBcAghind-as to generate the full-length gene. The full length gene was then digested with EcoRI (GAATTC) and HindIII (AAGCTT) enzymes and cloned into the pKK vector (Pharmacia) cut at the same restriction sites.

Example 32

Blocking free cysteine residues of HBcAg and then crosslinking

The free cysteine residue of HBcAg-Lys prepared as described above in example 23 was blocked with iodoacetamide. The blocked HBcAg-Lys was then cross-linked to the FLAG peptide using the heterobifunctional cross-linker m-maleimidobenzoyl-N-hydroxysuccinimide ester (Sulfo-MBS).

The method for blocking free cysteine residues and cross-linking HBcAg-Lys is as follows. HBcAg-Lys (550. mu.g/ml) was reacted with iodoacetamide (Fluka Chemie, Brugg, Switzerland) at a concentration of 50mM in Phosphate Buffer (PBS) (50mM sodium phosphate, 150mM sodium chloride, pH7.2) in a total volume of 1ml for 15 minutes at room temperature. The HBcAg-Lys thus modified was immediately reacted with 330. mu.M of Sulfo-MBS (Pierce) directly in the reaction mixture of step 1 at room temperature for 1 hour. The reaction mixture was then cooled on ice and dialyzed against 1000 volumes of PBS pH 7.2. The dialyzed reaction mixture was finally reacted with 300. mu.M FLAG peptide (CGGDYKDDDDK (SEQ ID NO: 147) containing a free cysteine at the N-terminus for coupling to activated HBcAg-Lys and applied to SDS-PAGE for analysis.

The pattern of bands obtained on the SDS-PAGE gel showed another clear band, which migrated slower than the control HBcAg-Lys derivatized with cross-linker but not reacted with the FLAG peptide. Without pre-blocking the cysteine with iodoacetamide, the HBcAg-Lys monomer was fully cross-linked to form a higher molecular weight molecule under the same conditions.

Example 33

Separation of Escherichia coli type 1 pili and chemical coupling with FLAGE peptides using heterobifunctional cross-linking agents

A. Introduction to the word

Bacterial pili or pili are filamentous surface organelles produced by a variety of bacteria. These organelles mediate the attachment of bacteria to host cell surface receptors, which is required for the development of a variety of bacterial infections such as cystitis, pyelonephritis, neonatal meningitis and diarrhea.

Pili can be classified into different categories according to receptor specificity (agglutination of blood cells from different species), assembly pathways (exocytosis, general secretion, chaperone/homing (usher), alternative chaperone) and morphological characteristics (rigid rough pili; flexible pili; atypical structures, including bursa; frizzled pili; etc.). Examples of rigid crude pili that form right-handed helices that assemble and mediate adhesion to host glycoproteins via the so-called chaperone/leader pathway include type 1 pili, P-pili, S-pili, F1C-pili, and 987P-pili. The most prominent and characteristic members of this class of pili are the P-pili and type 1 pili (reviewed in Soto, G.E. & Hultgren, S.J., J.Bacteriol.181: 1059-.

Type 1 pili are long filamentous polyprotein structures on the surface of escherichia coli. They have adhesive properties and bind to mannose-containing receptors present on the surface of a particular host tissue. 70-80% of all E.coli isolates express type 1 fimbriae, and one E.coli cell can carry up to 500 fimbriae. Type 1 pili typically reach a length of 0.2-2 μ M, average number of 1000 protein subunits, combined into a right-handed helix, 3.125 subunits per turn, 6-7nm in diameter, and 2.0-2.5nm in the central pore.

The major component FimA of type 1 pili accounts for 98% of the total pilus protein and is a 15.8kDa protein. The minor pilus components FimF, FimG and FimH are incorporated at the tip at a fixed distance along the pilus axis (Klemm, P. & Krogfelt, k.a., "pili type 1 of e.coli", "pili", Klemm, P. eds., CRC Press inc., (1994) pp.9-26). FimH is a 29.1kDa protein that is shown to be mannose-binding adhesin of type 1 pili (Krogfelt, k.a. et al, infection.immun.58: 1995-. It has recently been shown that FimH may also form a fine cilium at the end of the pilus (Jones, c.h. et al, proc.natl.acad.sci.usa 92: 2081-. The order of the major and minor components of the different mature pili is very similar, indicating a highly ordered assembly process (Soto, G.E. & Hultgren, S.J., J.Bacteriol.181: 1059-.

The P-pili of E.coli have a very similar structure with a diameter of 6.8nm, an axial hole of 1.5nm and 3.28 subunits per turn (Bullitt & Makowski, Biophys. J.74: 623; 632 (1998)). PapA at 16.6kDa is the major component of this pilus and shows 36% sequence identity and 59% similarity to FimA (see Table 1). As in type 1 pili, 36.0 kDaP-pilin adhesin PapG and the specialized adaptor protein account for only a very small fraction of the total pilin protein. The most obvious difference from type 1 pili is that adhesin is not part of the pilus shaft, and that it is located only in the terminal pili, which are linked to the pilus shaft by specialized adaptor proteins not found in type 1 pili (Hultgren, S.J. et al, Cell 73: 887-901 (1993)).

Table 1: similarity and identity (percentage) between several structural pilin proteins of type 1 pili and P-pili. The adhesins were ignored.

Similarity of characters

FimA PapA FimI FimF FimG PapE PapK PapH PapF

FimA 59 57 56 44 50 44 46 46

PapA 36 49 48 41 45 49 49 47

FimI 35 31 56 46 40 47 48 48

Same as FimF 3426304047434948

A FimG 2828282639394145

Sex PapE 2523182822434754

PapK 24 29 25 28 22 18 49 53

PapH 22 26 22 22 23 24 23 41

PapF 18 22 22 24 28 27 26 21

Type 1 pili are very stable hetero-oligomeric complexes. Neither SDS treatment nor protease digestion, boiling or addition of denaturants can separate type 1 pili into individual protein components. It was initially found that a combination of different methods, such as incubation at 100 ℃ and pH1.8, can disaggregate and isolate the components (Eshdat, Y. et al, J.Bacteriol.148: 308-. Interestingly, type 1 fimbriae showed a tendency to break at the FimH incorporation site after mechanical agitation, resulting in fragments that displayed FimH adhesins at their tips. This can be explained by a mechanism by which bacteria shear pili to effective length under mechanical stress (Klemm, P. & Krogfelt, K.A., "pili type 1 of E.coli". pili, Klemm, P. eds., CRCPRess Inc. (1994) pp.9-26). Although they are particularly stable, type 1 pili are partially broken apart in the presence of 50% glycerol; they lose their helical structure and form an elongated, flexible, 2 nm-wide protein chain (Abraham, S.N. et al, J.Bacteriol.174: 5145-.

The P-pili and type 1 pili are encoded by a single gene cluster on the chromosome of E.coli of approximately 10kb (Klemm, P & Krogfelt, K.A., "type 1 pili of E.coli" -pili ", Klemm, P. eds., CRC Press Inc. (1994) pp.9-26; Orndorff, P.E. & Falkow, S., J.bacteriol.160: 61-66 (1984)). A total of 9 genes were found in the type 1 pilus gene cluster and 11 genes were found in the P-pilus cluster (Hultgren, S.J. et al, adv.prot.chem.44: 99-123 (1993)). The composition of these two clusters is very similar.

The first two fim genes, fimB and fimE, encode recombinases involved in the regulation of pilus expression (McClain, M.S. et al, J.Bacteriol.173: 5308-5314 (1991)). The major structural pilin proteins are encoded by the next gene fimA of the cluster (Klemm, P., Euro.J.biochem.143: 395-400 (1984); Orndorff, P.E. & Falkow, S., J.Bacteriol.160: 61-66 (1984); Orndorff, P.E. & Falkow, S., J.Bacteriol.162: 454-457 (1985)). The exact role of fimI is not clear. It has been reported to be incorporated into pili as well (Klemm, P. & Krogfelt, K.A., "pili type 1 of E.coli". pili, Klemm, P. eds., CRC Presssen., (1994) pp.9-26). The adjacent fimC does not encode a structural component of mature pili, but rather encodes the so-called pili chaperone proteins necessary for pili assembly (Klemm, P., Res. Microbiol.143: 831-.

The assembly platform in the outer membrane of mature pili-anchored bacteria is encoded by fimD (Kleemm, P. & Christiansen, g., mol. gen. genetics 220: 334-. The three minor components of type 1 FimF, FimG and FimH are encoded by the last three genes of the cluster (Klemm, P. & Christiansen, g., mol. gen. genetics 208: 439-445 (1987)). All genes, except fimB and fimE, encode precursor proteins that are secreted into the periplasm via the secretory pathway.

The similarity between different pili after the chaperonin/leader pathway is not limited to their morphological characteristics. Their genes are also arranged in a very similar manner. The gene encoding the major structural subunit is usually located directly downstream of the regulatory element at the beginning of the gene cluster, followed by the gene encoding the other structural subunit (fimI for type 1 pili, papH for P-pili). PapH was shown to stop pilus assembly, and it was concluded that FimI could do so (Hultgren, S.J. et al, Cell 73: 887-. Two proteins that direct the pilus formation process, namely a specialized pilin chaperone protein and an outer membrane assembly platform, are located adjacently downstream. At the end of the gene cluster, an indefinite number of minor pilus components, including adhesins, are encoded. The similarity in morphological structure, sequence (see table 1), genetic organization and regulation suggests a close evolutionary relationship and a similar assembly process for these organelles.

Bacteria producing type 1 fimbriae exhibit so-called phase transformation. The bacteria are completely fimbriae or are sterile. This is achieved by inverting the 314bp genomic DNA fragment containing the fimA promoter, thereby inducing "all-on" or "all-off expression of the pilus gene (McClain, M.S. et al, J.Bacteriol.173: 5308-. The coupling of the expression of other structural pilus genes to the fimA expression is achieved by a mechanism which is still unknown. However, a number of studies have elucidated mechanisms that influence the switching of these two phenotypes.

The first two genes fimB and fimE of the type 1 fimbriae gene cluster encode recombinases that recognize a two-fold symmetric 9bpDNA fragment flanked by invertible fimA promoters. FimB switches pili to "on" and FimE turns the promoter on in the "off" direction. Thus, positive or negative regulation of fimB or fimE expression controls the so-called "fim-switch" position (McClain, M.S. et al, J.Bacteriol.173: 5308-.

The two regulatory proteins fimB and fimE are transcribed from different promoters, their transcription being affected by a number of different factors, including Integrating Host Factor (IHF) (Blomfield, I.C. et al, mol. Microbiol.23: 705-717(1997)) and leucine-reactive regulatory protein (LRP) (Blomfield, I.C. et al, J.Bacteriol.175: 27-36 (1993); Gally, D.L. et al, J.Bacteriol.175: 6186-6193 (1993); Gally, D.L. et al, Microbiol.21: 725-738 (1996); Roesch, R.L. & Blomfield, I.C., mol. Microbiol.27: 751-761 (1998)). Mutations in the former lock the bacteria into an "on" or "off" state, while the frequency of LRP mutant turnover is reduced. The effect of leuX on pilus biogenesis is also shown. The gene is located near the fim gene on the chromosome and encodes the minor leucine tRNA (minor leucine tRNA) of the UUG codon. fimB contains 5 UUG codons, whereas fimE contains only two, and increased leuX transcription will favor fimB expression over fimE expression (Burghoff, R.L. et al, infection. Immun.61: 1293-882 (1993); Newman, J.V. et al, FEMSMICrobiol. Lett.122: 281-287 (1994); Ritter, A. et al, mol. Microbiol.25: 871-882 (1997)).

In addition, temperature, medium composition and other environmental factors can also affect the activity of FimB and FimE. Finally, spontaneous, statistical switching of the fimA promoter has been reported. The frequency of this spontaneous conversion is about 10 per generation^-3(Eisenstein, B.I., Science 214: 337-339 (1981); Abraham, S.M. et al, Proc. nat. Acad. Sci, USA 82: 5724-5727(1985)), but is strongly influenced by the above factors.

Genes fimI and fimC were also transcribed from the fimA promoter, but a DNA fragment was identified directly downstream of fimA, which has a strong tendency to form secondary structures, possibly a partial transcription terminator (Klemm, P., Euro.J. biochem.143: 395-E400 (1984)); it is therefore assumed that it severely reduces transcription of fimI and fimC. At the 3' end of fimC, another promoter controls fimD transcription; at the 3' end of fimD there is a newly known fim promoter, which regulates the levels of FimF, fimG and FimH. Thus, all minor type 1 pilin proteins are transcribed as an mRNA (Klemm, P. & Krogfelt, K.A., "type 1 pili of E.coli". pili ", Klemm, P. eds., CRC Press Inc. (1994) pp.9-26). This ensures a 1: 1 stoichiometry at the mRNA level, which is likely to be maintained at the protein level.

For P-pili, we found an additional regulatory mechanism when determining the half-life of mRNA of different P-pili genes. The mRNA of papA is very long-lived, whereas the mRNA of papB, a regulatory pilin, is encoded by short-lived mRNAs (Naureckien, S. & Uhlin, B.E., mol. Microbiol.21: 55-68 (1996); Nilsson, P. et al, J. bacteriol.178: 683- "690 (1996)).

For type 1 pili, the gene for the type 1 pilus chaperone FimC begins at the GTG but not at the ATG codon, resulting in a decrease in translation efficiency. Finally, analysis of fimH gene showed that fimHmRNA has a tendency to form stem-loop, which may seriously hinder translation. In summary, the biogenesis of bacterial pili is regulated by a number of different mechanisms that act at all levels of protein biosynthesis.

Periplasmic pilins are usually synthesized as precursors, containing an N-terminal signal sequence that allows transport through the inner membrane using secretory apparatus. After transport, the precursor is usually cleaved by signal peptidase I. Type 1 pilus structural subunits typically contain disulfide bonds, their formation being catalyzed by DsbA and possibly by DsbC and DsbG gene products.

Type 1 FimC, a FimC protein, lacks cysteine residues. In contrast, the chaperone protein PapD of P-pili is the only disulfide bond-containing member of the pili chaperone family, and P-pili dependence on DsbA has been determined (Jacob-Dubuisson, F. et al, Proc. Nat. Acad. Sci. USA 91: 11552-11556 (1994)). PapD does not accumulate in the periplasm of the Δ dsbA strain, indicating that the lack of chaperone protein results in a hampered P-pilus assembly mechanism (Jacob-Dubuisson, F. et al, Proc. Nat. Acad. Sci. USA 91: 11552-11556 (1994)). This is consistent with the following findings: type 1 fimbriae still assemble but at reduced levels in the Δ dsbA strain (Hultgren, s.j. et al, "bacterial adhesion and assembly thereof", "escherichia coli and salmonella", Neidhardt, f.c., eds., ASM Press, (1996) pp.2730-2756).

Type 1 pili and P-pili 98% consist of single or major structural subunits called FimA and PapA, respectively. Both proteins were 15.5kDa in size. The other minor components encoded by the two pilus gene clusters were very similar (see table 1). The similarity in sequence and size of the other subunits, except adhesin, suggests that they all share the same folding motif, but differ in affinity to each other. In particular, the N-and C-terminal regions of these proteins are well conserved, presumably playing an important role in chaperonin/subunit interactions and subunit/subunit interactions within the pilus (Soto, G.E. & Hultgren, S.J., J.Bacteriol.181: 1059-1071 (1999)). Interestingly, conserved N-terminal fragments can be found in the middle of the pilus adhesins, suggesting that the adhesins have two structural domains, where the C-terminal domain starting from the conserved motif corresponds to the structural pilus subunit, while the N-terminal domain is responsible for the recognition of the host cell receptor (Hultgren, S.J. et al, Proc. Nat. Acad. Sci. USA 86: 4357-4361 (1989); Haslam, D.B. et al, mol. Microbiol.14: 399-409 (1994); Soto, G.E. et al, EMBO J.17: 6155-6167 (1998)). Different subunits have also been shown to affect the morphological characteristics of pili. It has been reported that the removal of several genes reduces the number of type 1 or P-pili or increases their length (fimH, papG, papK, fimF, fimG) (Russell, P.W. & Orndorff, P.E., J.Bacteriol.174: 5923-; the combination of gene deletions amplifies this effect or leads to a complete failure to form pili (Jacob-Dubuisson, R. et al, EMBO J.12: 837-847 (1993)).

The conserved C-terminal region differs in non-pilus-adherent cellular organelles, such as Myf pili and CS3 pili, which are also assembled by chaperonin/leader systems. This indirectly demonstrates the importance of these C-terminal subunit fragments for quaternary structural interactions (Hultgren, S.J. et al, "bacterial adhesion and assembly thereof", "E.coli and Salmonella", Neidhardt, F.C. eds., ASMPress, (1996) pp.2730-2756).

Gene deletion studies have demonstrated that removal of the pilo chaperone protein results in complete inability to form P-pili and type 1 pili (Lindberg, F. et al, J. bacteriol.171: 6052-. Periplasmic extracts of the Δ fimC strain showed major subunit FimA aggregation, but no pili could be detected (Klemm, p., res. microbiol.143: 831-. Attempts to overexpress a single P-pilus subunit failed, and only the proteolytic form could be detected in the absence of PapD; in addition, P-pilin was purified from the inner membrane fraction in the absence of chaperone proteins (Lindberg, F. et al, J.Bacteriol.171: 6052-6058 (1989)). However, for the FimC/FimH complex and the different Pap-proteins, including the adhesin PapG and the major subunit PapA, co-expression of the structural pilin protein and its chaperone protein enables the chaperone protein/subunit complex to be detected in the periplasm (Tewari, R. et al, J.biol. chem.268: 3009-3015 (1993); Lindberg. F. et al, J.Bacteriol.171: 6052-6058 (1989)). The affinity of chaperonin/subunit complexes for their assembly platform was also studied in vitro and found to be very different (Dodson et al, Proc Natl. Acad. Sci. USA 90: 3670-3674 (1993)). From these results, it was suggested that the pilin chaperone protein had the following functions.

They are assumed to recognize unfolded pilus subunits, preventing them from clumping, providing a "folding template" that directs the formation of native structures.

The folded subunits exhibit a surface that allows subunit/subunit interaction after folding, which is expected to be protected from interaction with other subunits and remain in a monomeric, assemblable state.

Finally, it is speculated that the pilin chaperone protein triggers the release of subunits at the outer membrane assembly site, affecting the composition and order of mature pili by triggering with different efficiencies (see below for separate sections).

After release of the subunits on the outer membrane, the chaperones are in a free state in order to participate in another round of substrate binding, aid folding, transport of the subunits through the periplasm and specific transport to the assembly site. Due to the periplasm's lack of energy sources, such as ATP, the entire pilus assembly process must be thermodynamically driven (Jacob-Dubuisson, F. et al, Proc Natl. Acad. Sci. USA 91: 11552-. The diverse functions of pilin chaperones involve a very finely tuned cascade of steps.

However, there are several findings that cannot be easily explained by the above-mentioned functional model of pilin chaperone proteins. An example is the presence of a multimeric chaperonin/subunit complex (Striker, R.T. et al, J.biol.chem.269: 12233-12239(1994)) in which a chaperonin binds to a dimer or trimer of subunits. It is difficult to imagine a folding template that can be "double utilized". Studies of molecular details on chaperonin/subunit interactions (see below) partially support the above-described functions, but new problems also arise.

To date, all 31 periplasmic chaperones identified by genetic studies or sequence analysis are proteins of about 25kDa with very high pI values around 10. 10 of these chaperones help the assembly of the rod pili, 4 are involved in the formation of the pili, 10 are important for the biogenesis of atypical fine structures, including capsule-like structures, and 2 adhesion structures have not been determined so far (Holmgren, A. et al, EMBO J.11: 1617-. The pairwise sequence identity between these chaperones and PapD is 25-56%, indicating overall fold identity (Hung, D.L. et al, EMBO J.15: 3792-.

The first study on chaperonin/substrate recognition was based on the finding that the C-termini of all known pilin proteins are very similar. ELISA assays showed that the synthetic peptide corresponding to the C-terminus of P-pilin bound to PapD (Kuehn, M.J. et al, Science 262: 1234-1241 (1993)). Most importantly, the X-ray structure of both complexes was resolved in which PapD co-crystallized with a 19 residue peptide corresponding to the C-terminus of the adhesin PapG or the minor pilus component PapK (Kuehn, M.J. et al, Science 262: 1234-. Both peptides bind to the beta-strand of the chaperonin N-terminal domain towards the gap of the domain in an extended conformation, thus elongating the beta-sheet with the other strand. The C-terminal carboxylic acid group of the peptide is anchored to Arg8 and Lys112 by hydrogen bonds, these two residues being constant in the pilin chaperone family. Mutagenesis studies confirm their importance, as their exchange with alanine leads to the accumulation of nonfunctional pilin chaperones in the periplasm (Slonim, L.N. et al, EMBO J.11: 4747-4756 (1992)). The crystal structure of PapD indicates that neither Arg8 nor Lys112 is involved in chaperone stabilization, but that exposure to solvent is complete (Holmgren, A. & Branden, C.I., Nature 342: 248-251 (1989)). It is reported that exchange of the C-terminal PapA residue on the substrate prevents P-pilus formation, and similar experiments on a conserved C-terminal fragment of P-pilus adhesin PapG prevent its incorporation into P-pilus (Hultgren, s.j. et al, "bacterial adhesion and assembly thereof", "escherichia coli and salmonella", Neidhardt, f.c. eds, ASM Press, (1996) pp.2730-2756). Thus, all evidence suggests that the pilus subunit is recognized by the C-terminal fragment of the subunit.

A recent study on the C-terminal amino acid exchange of P-pili adhesin PapG gives more detailed information. Amino acid substitutions at positions-2, -3, -6, -8 relative to the C-terminus are tolerated, but alter pilus stability (Soto, G.E. et al, EMBO J.17: 6155-6167 (1998)).

However, when this model is studied more carefully, some problems arise. Adherent bacterial structures that do not assemble into rigid, rod-shaped pili lack conserved C-terminal fragments (Hultgren, s.j. et al, "bacterial adhesion and assembly thereof", e.coli and salmonella, Neidhardt, f.c. eds, ASMPress, (1996) pp.2730-2756), although they also depend on the presence of the relevant pili chaperone proteins. This suggests that the C-terminal fragment of the pilus subunit has a different role, i.e. mediation of quaternary structure interactions in the mature pilus. Furthermore, attempts to resolve the structure by NMR were severely hampered due to the weak binding of the C-terminal peptide to chaperones in the chaperone complex (Walse, B. et al, FEBS Lett.412: 115-120 (1997)); while one major contribution of the C-terminal fragment to chaperone recognition is the relatively high affinity effect.

Another problem arises if variability between different subunits is taken into account. Although the C-terminal fragment is conserved, a number of conservative substitutions are found. For example, 15 of the 19 amino acid residues differ between two peptides co-crystallizing with PapD (Soto, G.E. et al, EMBO J.17: 6155-6167 (1998)). This has been explained by the type of interaction between chaperones and substrates, which occurs primarily through backbone interactions, rather than specifically through side chain interactions. Moreover, the specificity of chaperones for certain substrates is not readily explained. Contrary to the foregoing, conserved residues have been considered evidence of specificity (Hultgren, S.J. et al, "bacterial adhesion and assembly thereof", "E.coli and Salmonella," Neidhardt, F.C. eds., ASM Press, (1996) pp.2730-2756).

The outer membrane assembly platform, also known in the literature as "leader" (usher), is formed from homo-oligomers of FimD or PapC in type 1 and P-pilus, respectively (Klemm, P. & Christiansen, G., mol. Gen. genetics 220: 334-. An examination of the elongation of type 1 pili by electron microscopy confirmed that the pili elongated from the bottom (Lowe, M.A. et al, J.Bacteriol.169: 157-163 (1987)). Unlike secretion of unfolded subunits into the periplasmic space, fully folded proteins must be transferred across the outer membrane, possibly in an oligomeric form (Thanassi, D.G. et al, Proc. Nat. Acad. Sci. USA 95: 3146-. This firstly requires a sufficiently wide membrane pore to allow passage and secondly a thermodynamically driven transport mechanism (Jacob-Dubuisson, F. et al J.biol.chem.269: 12447-12455 (1994)).

FimD expression alone has been shown to be detrimental to bacterial growth, and co-expression of pilus subunits has been shown to restore normal bacterial growth (Klemm, P. & Christiansen, g., mol. gen. genetics 220: 334-. It was concluded that the leader protein may form a well that is completely filled with pili. Observation of the PapC-bound membrane vesicles by electron microscopy confirmed the presence of a pore-forming structure with an internal diameter of 2nm (Thanassi, D.G. et al, Proc. nat. Acad. Sci. USA 95: 3146-3151 (1998)). Since the inner diameter of the pores is too small, the pilus rods cannot pass through, suggesting that a helical arrangement of mature pili is formed outside the bacterial surface. Glycerol was found to break down the pili and then form an approximately 2nm protein chain, which was well matched to this hypothesis, since an extended subunit chain could be formed in the well as the first step (Abraham, S.N. et al, J.Bacteriol.174: 5145-. The formation of spiral pilus rods outside the bacterial membrane may be the driving force for the transfer of the growing pili through the membrane.

It has also been shown that the leaders of type 1 and P-pili form ternary complexes that bind chaperonin/subunit complexes with different affinities (Dodson, K.W. et al, Proc. Nat. Acad. Sci. USA 90: 3670- > 3674 (1993); Saulino, E.T. et al, EMBO J.17: 2177- > 2185 (1998)). This can be interpreted as a "kinetic assignment" of the pilin proteins in a defined order in the pili. It is also proposed that the structural protein may present a binding surface which is compatible only with another type of pilin; this would be another mechanism for obtaining highly fixed order subunits in mature fimbriae (Saulino, E.T. et al, EMBO J.17: 2177-.

B. Preparation of Escherichia coli type 1 pili

Escherichia coli W3110 strain was spread on LB (10g/L tryptone, 5g/L yeast extract, 5g/L NaCl, pH7.5, 1% agar (W/v)) plate, and incubated overnight at 37 ℃. Then 5ml of LB starter was inoculated with a single colonyCulture medium (10g/L tryptone, 5g/L yeast extract, 5g/L NaCl, pH 7.5). After 24 hours of incubation under conditions favoring the production of type 1 pili by the bacteria (37 ℃ C., without stirring), 5 flasks containing 1 liter of LB were each inoculated with 1ml of the initial culture broth. The bacterial culture was then incubated at 37 ℃ for a further 48-72 hours without stirring. The bacteria were collected by centrifugation (5000rpm, 4 ℃, 10 min) and the resulting pellet was resuspended in 250ml 10mM Tris/HCl, pH 7.5. The pili were separated from the bacteria in a common mixer with stirring at 17000rpm for 5 minutes. After centrifugation at 10000rpm for 10 minutes at 4 ℃, the pilus-containing supernatant was collected and 1M MgCl was added ₂To a final concentration of 100 mM. The solution was allowed to stand at 4 ℃ for 1 hour, and then the pili were sedimented by centrifugation (10000rpm, 20 minutes, 4 ℃). The pellet was resuspended in 10mM HEPES, pH7.5, and then the residual cell debris was removed by a final centrifugation step to clarify the pilus solution.

C. Coupling of purified E.coli type 1 pili with m-maleimidobenzoyl-N-hydroxythiosuccinimide ester (sulfo-MBS) FLAG

600 μ l of a 95% pure bacterial type 1 pilus solution (2mg/ml) was incubated with the heterobifunctional crosslinker sulfo-MBS (0.5mM) for 30 minutes at room temperature. Subsequently, the mixture was dialyzed overnight against 1 liter of 50mM phosphate buffer (pH7.2) containing 150mM NaCl to remove free sulfo-MBS. Then 500. mu.l of derivatized pili (2mg/ml) were mixed with 0.5mM FLAG peptide (containing an amino-terminal cysteine) in the presence of 10m MEDTA in order to prevent metal-catalyzed thiol oxidation. The uncoupled peptides were removed by exclusion chromatography.

Example 34

Construction of expression plasmid for expressing E.coli type 1 pilus

The DNA sequence disclosed in GenBank accession No. U14003, the entire disclosure of which is incorporated herein by reference, contains all of the E.coli genes required for the production of type 1 pili, from nucleotide 233947 to nucleotide 240543(fim gene cluster). The partial sequence contains the sequences of the genes fimA, fimI, fimC, fimD, fimF, fimG and fimH. To amplify this portion of the E.coli genome, three different PCRs were performed as described below, followed by cloning into pUC19(GenBank accession Nos. L09137 and X02514).

PCR templates were prepared as follows: a10 ml glycerol stock solution of Escherichia coli W3110 strain was mixed with 90ml water, the mixture was boiled at 95 ℃ for 10 minutes, and then centrifuged at 14000rpm for 10 minutes in a tabletop centrifuge, and the supernatant was collected.

10ml of the supernatant was mixed with 50pmol of PCR primer one and 50pmol of PCR primer two as described below. 5ml of 10 XPCR buffer, 0.5ml of Taq-DNA-polymerase and water are added to a total of 50 ml. All PCR was performed according to the following protocol: 2 minutes at 94 ℃ followed by 30 cycles of 20 seconds at 94 ℃, 30 seconds at 55 ℃ and 2 minutes at 72 ℃. The PCR product was then purified by 1% agarose gel electrophoresis.

The sequence from nucleotide 233947 to nucleotide 235863 was amplified with oligonucleotides having the following sequence, including the fimA, fimI and fimC genes:

TAGATGATTACGCCAAGCTTATAATAGAAATAGTTTTTTGAAAGGAAAGCAGCATG (SEQ ID NO: 196) and

GTCAAAGGCCTTGTCGACGTTATTCCATTACGCCCGTCATTTTGG(SEQID NO：197)

both oligonucleotides also contained flanking sequences that allowed the amplification product to be cloned into pUC19 by restriction enzyme sites HindIII and SaII. The resulting plasmid was designated pFIMIC (SEQ ID NO: 198).

The sequence from nucleotide 235654 to nucleotide 238666 was amplified with oligonucleotides having the following sequence, including the fimD gene:

AAGATCTTAAGCTAAGCTTGAATTCTCTGACGCTGATTAACC (SEQ ID NO: 199) and

ACGTAAAGCATTTCTAGACCGCGGATAGTAATCGTGCTATC(SEQ IDNO：200)。

both oligonucleotides also contained flanking sequences that allowed the amplification product to be cloned into pUC19 by restriction enzyme sites HindIII and XbaI. The resulting plasmid was designated pIMD (SEQID NO: 201).

The sequence from nucleotide 238575 to nucleotide 240543 was amplified with oligonucleotides having the following sequences, including fimF, fimG and fimH genes:

AATTACGTGAGCAAGCTTATGAGAAACAAACCTTTTTATC (SEQ ID NO: 202) and

GACTAAGGCCTTTCTAGATTATTGATAAACAAAAGTCACGC(SEQ IDNO：203)。

both oligonucleotides also contained flanking sequences that allowed the amplification product to be cloned into pUC19 by restriction enzyme sites HindIII and XbaI. The resulting plasmid was designated pFIFGH (SEQ ID NO: 204).

The following cloning procedure was then performed to generate plasmids containing all of the fim genes described above.

pFIMIC was digested with EcoRI and HindIII (2237-. The fragments were then ligated and the resulting plasmid containing all fim genes required for pilus formation was designated pFIMICDFGH (SEQ ID NO: 205).

Example 35

Construction of an expression plasmid for expression of E.coli type 1 pili lacking adhering FimH

The plasmid pFICIDFGH (SEQ ID NO: 205) was digested with KpmI, followed by 0.7% agarose gel electrophoresis to separate the fragment consisting of nucleotides 8895-8509 and circularization by self-ligation. The resulting plasmid, designated pFIMICDFG (SEQ ID NO: 206), lacks the fimH gene and can be used to produce fiMH-free type 1 fimbriae.

Example 36

Expression of type 1 pilus using plasmid pFICIDFGH

Escherichia coli W3110 strain was transformed with pFICIDFGH (SEQ ID NO: 205), spread on LB (10g/L tryptone, 5g/L yeast extract, 5g/L NaCl, pH7.5, 1% agar (W/L agar) containing 100. mu.g/ml ampicillinV)) plates were incubated overnight at 37 ℃. Then, 50ml of LB-glucose starting medium (10g/L tryptone, 5g/L yeast extract, 1% (w/v) glucose, 5g/L NaCl, pH7.5, 100mg/ml ampicillin) was inoculated with a single colony. After incubation at 37 ℃ for 12-16 hours at 150rpm, a 5 liter shake flask containing 2 liters of LB-glucose was inoculated with 20ml of the starting broth. The bacterial culture was then incubated at 37 ℃ for a further 24 hours with stirring (150 rpm). The bacteria were collected by centrifugation (5000rpm, 4C, 10 min) and the resulting pellet was resuspended in 250ml 10mM Tris/HCl, pH 8. The pili were separated from the bacteria in a common mixer with stirring at 17000rpm for 5 minutes. After centrifugation at 10000rpm for 10 minutes at 4 ℃, the pilus-containing supernatant was collected and 1M MgCl was added ₂To a final concentration of 100 mM. The solution was allowed to stand at 4 ℃ for 1 hour, and then the precipitated pili were centrifuged (10000rpm, 20 minutes, 4 ℃). The pellet was resuspended in 10mM HEPES, 30mM EDTA, pH7.5, at room temperature for 30 minutes, and then the residual cell debris was removed by a final centrifugation step to clarify the pilus solution. The resulting preparation was then dialyzed against 20mM HEPES, pH 7.4.

Example 37

Conjugation of IgE epitopes and mimotopes to E.coli type 1 pili

Mu.l aliquots of 100. mu.M solution of the heterobifunctional crosslinker, sulfa-MBS, were added to 400. mu.l 95% pure bacterial type 1 pilus solution (2.5mg/ml, 20mM HEPES, ph7.4) followed by 45 minutes incubation at room temperature with stirring. Thereafter, excess of the sulpho-MBS was removed by size exclusion chromatography using a PD-10 column. In addition, the cross-linking agent can also be removed by dialysis. To a 1ml aliquot of the derivative pilus (1-1.25mg/ml, 20mM HEPES pH7.4) was added 1.3. mu.l of a solution containing 1.1mg/ml of peptide Ce3epi (CGGVNLTWSRA SG (SEQ ID NO: 207)) or peptide Ce3Mim (CGGVNLPWSFGLE (SEQ ID NO: 208)). The samples were incubated at room temperature for 4 hours and dialyzed 2 times against 2L of buffer containing 20mM HEPES (pH7.4) to remove the unconjugated peptide. Furthermore, the unconjugated peptide may be removed by size exclusion chromatography.

Example 38

Using melittin coupled to Q beta-capsid proteinEsterase A₂(PLA₂) Fusion protein immunization of mice

A. Preparation of PLA for cytoplasmic expression₂Alternative vectors for the catalytically inactive variant of a gene fused to the amino acid sequence AAASGGCGG (SEQ ID NO: 209)

The PLA of example 9 was amplified by PCR from pAV3PLAfos using the oligonucleotides ecori _ Ndel _ PLA (see below for sequence) and PLA-Cys-hind (example 29)₂A gene construct. For the PCR reaction, 50. mu.l of a solution containing 1.2 units of PfxDNA polymerase (Gibco), 1mM MgSO₄And 200 u M dNTPs and 10 times diluted Pfx enhancing solution (Gibco) in the reaction mixture using 100pmol each primer and about 1 u g PAV3PLAFOs DNA. The temperature cycle of the reaction was carried out as follows: 94 ℃ for 2 min, 92 ℃ (0.5 min), 58 ℃ (0.5 min), 68 ℃ (1 min) for 5 cycles; 92 deg.C (0.5 min), 63 deg.C (0.5 min), 68 deg.C (1 min) for 25 cycles. The PCR product was purified by agarose gel electrophoresis, followed by isolation of the fragment using the Qiagen Qiaquick kit, digestion with the enzymes NdeI and HindIII, and cloning into the PET11a vector (Novagen) digested with the same enzymes.

Oligonucleotide: ecori _ Ndel _ pla

TAACCGAATTCAGGAGGTAAAAACATATGGCTATCATCTACC(SEQ IDNO：214)

The vector encodes a fusion protein having the following amino acid sequence:

MAIIYPGTLWCGHGNKSSGPNELGRFKHTDACCRTQDMCPDVMSAG

ESKHGLTNTASHTRLSCDCDDKFYDCLKNSADTISSYFVGKMYFNLIDTK

CYKLEHPVTGCGERTEGRCLHYTVDKSKPKVYQWFDLRKYAAASGGCGG(SEQ ID NO：210)

PLA₂conjugation of fusion proteins to Q β capsid proteins

600 μ L Q β capsid protein solution (2mg/ml in 20mM Hepes, pH7.4) was reacted with 176 μ L Sulfo-MBS (13mg/ml aqueous solution) for 60 minutes at room temperature and dialyzed against 1L20mM Hepes pH 7.4O/N at 4 ℃. The next day500. mu.l of PLA2 solution (2.5mg/ml) containing 0.1mM DTT was desalted through a 5ml Hi-Trap column (Pharmacia). Reduced and desalted PLA₂(60. mu.l of about 0.5mg/ml solution) was mixed with activated dialyzed Q.beta.capsid (25. mu.l of 1.5mg/ml solution) and reacted at room temperature for 4 hours.

In association with pLA₂The capsid with a diameter of 25-30nm is clearly visible on electron microscope images of the Q β capsid protein taken before and after coupling.

C. With PLA coupled to Q.beta.capsid protein₂Immunization of mice

Female Balb/c mice received 50 ug of PLA on day 0₂The coupled Q β capsid proteins were immunized intravenously, boosted with the same amount of antigen on day 14. Blood was taken from the mice on day 20 and sera were analyzed by ELISA. Obtaining a 1: 5000 PLA resistance₂The titer.

Example 39

Conjugation of IgE mimotopes and epitopes to Q β capsid proteins

The N-terminal cysteine residue was used to couple the human IgE epitope with the following amino acid sequence to the Q β capsid protein:

ce3 epitope: CGGVNLTWSRASG (SEQ ID NO: 207)

Ce3 mimics bit: CGGVNLPWSFGLE (SEQ ID NO: 208)

Coupling reactions were performed with Sulfo-MBS activated Q.beta.capsid proteins followed by dialysis to remove excess crosslinker. The epitope or the mimotope was diluted into the reaction mixture containing the activated Q β capsid, respectively, and reacted at room temperature for 4 hours. Finally, the reaction mixture was dialyzed against PBS for 4 hours and injected into mice.

The following cyclic mimotopes are also coupled to Q β capsid protein:

ce4 mimics bit: GEFCINHRGYWVCGDPA (SEQ ID NO: 211).

To introduce a protected thiol group into the mimic site, the mimic site is first reacted with the chemical group N-succinimide-S-acetylthioacetic acid (SATA). Then treated with hydroxylamine to remove the protecting group and reacted immediately with the activated Q β capsid protein for 4 hours at room temperature. Finally, the reaction mixture was dialyzed for 4 hours and injected into mice.

Example 40

Immunization of mice with HBcAg-Lys coupled to M2 peptide

Coupling of M2 peptides to HBcAg-Lys capsid proteins

To elicit an immune response against the M2 peptide, a synthetic M2 peptide containing a cysteine residue at the C-terminus corresponding to the N-terminal fragment of influenza M2 protein

(SLLTEVETPIRNEWGCRCNGSSDGGGC (SFQ ID NO: 212)) was chemically coupled to purified HBcAg-Lys particles. Sulfo-MBS (232. mu.l, 3mM) was reacted with 1.4ml of HBcAg-Lys in PBS (1.6 mg/ml). The mixture was dialyzed overnight against Phosphate Buffered Saline (PBS). M2 peptide was diluted with DMSO to a concentration of 24 mg/ml; mu.l of this solution was diluted with 300. mu.l of PBS and 188. mu.l were added to 312. mu.l of dialyzed and activated HBcAg-Lys solution. EDTA (10. mu.l of a 1M solution) was added to the reaction mixture, and the reaction was continued at room temperature for 4 hours.

Immunization of mice with HBcAg-Lys coupled to M2 peptide

Female Balb/c mice were immunized intravenously with 50 μ g HBcAg-Lys-M2 or M2 peptide alone on day 0 and boosted with the same amount of antigen 10 days later. After another 10 days, mice were infected intranasally with influenza virus (50pfu, PR/8) and the survival of infected mice was monitored. In addition, viral titers in the lungs were also determined. Mice pre-treated with the M2-HBcAg-Lys peptide were completely protected and virus was cleared by day 7.

EXAMPLE 41

Coupling of the M2 peptide to pili, Q β and cysteine-free HBcAg capsid protein, antibody titers obtained by immunization of mice with these coupled pili and capsids and of HBcAg1-183 with the M2 peptide

The titers obtained by immunizing mice with the N-terminal fusion protein were compared

Coupling of M2 peptides to pili, Q β and cysteine-free HBcAg-capsid proteins

Qβ: 1ml of a solution of 1mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.2 was reacted with 93. mu.l of a 13mg/ml aqueous solution of Sulfo-MBS (Pierce) on a shaker at room temperature for 30 minutes. The reaction solution was then dialyzed overnight against 2L of 20mM Hepes, 150mM NaCl pH 7.2. The dialyzed reaction mixture was then reacted with 58.8. mu.l of a stock solution of 25mM M2 peptide (SEQ ID NO: 212) in DMSO on a shaker at room temperature for 4 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 2L of 20mM Hepes, 150mM NaCl pH 7.2.

Cysteine-free HBcAg: a solution of 0.8mg/ml cysteine-free HBcAg capsid protein (example 31) in PBS pH7.2 (1.25 ml) was reacted with 93. mu.l of a 13mg/ml aqueous solution of sulfo-MBS (Pierce) on a shaker at room temperature for 30 minutes. The reaction solution was then dialyzed overnight against 2L of 20mM Hepes, 150mM NaCl pH 7.2. The dialyzed reaction mixture was reacted with 58.8. mu.l of a stock solution of 25mM M2 peptide (SEQ ID NO: 212) in DMSO on a shaker at room temperature for 4 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 2L of 20m MHepes, 150mM NaCl pH 7.2.

Pilus: mu.l of a solution of 2.5 mg/ml pilin 20mM Hepes pH7.4 was reacted with 60. mu.l of an aqueous solution of 100mM Sulfo-MBS (Pierce) on a shaker at room temperature for 45 minutes. The reaction solution was desalted using a PD-10 column (Amersham-Pharmacia Biotech) and a second fraction of 500. mu.l of protein (approx. containing 1g of protein) eluted from the column was reacted with 58.8. mu.l of a stock solution of 25mM M2 peptide (SEQ ID NO: 212) in DMSO on a shaker at room temperature for 4 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 2L of 20mM Hepes, 150mM NaCl pH 7.2.

Gene fusion of M2 peptide and HBcAg1-183

M2 fusion with Hbc gene: such as Neirynck et al, Nature Medicine 5: 1157(1999), clone M2 at the N-terminus of Hbc. MD-HBc was expressed in E.coli and purified by gel chromatography. The presence of the M2 peptide at the N-terminus of M2-HBc was confirmed by Edman sequencing.

Immunization of mice:

female Balb/c mice were vaccinated without adjuvant with M2 peptide coupled to pili, Q β and cysteine-free HbcAg protein and M2 peptide fused to the Hbc immunogen gene. 35 μ g of each sample protein was injected intraperitoneally on day 0 and day 14. Mice were bled on day 27 and sera were analyzed by M2 peptide-specific ELISA.

ELISA

ELISA plates were coated with 10. mu.g/ml M2 peptide coupled to RNAse. The plates were blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Preimmune serum was also tested as a control. Control ELISA experiments performed on sera of mice immunized with irrelevant peptide crosslinked to Hbc or other vehicle showed that the antibodies detected were specific for the M2 peptide. The results are shown in fig. 27A and B.

Example 42

Coupling of angiotensin I and angiotensin II to Q beta and immunization of mice with Q beta-angiotensin vaccine

A. Coupling of angiotensin I and angiotensin II to Q beta capsid protein

The following angiotensin peptides were chemically synthesized: CGGDRVYIHPF ("Angio I"), CGGDRVYIHPFHL ("Angio II"), DRVYIHPFHLGGC ("Angio III"), CDRVYIHPFHL ("Angio IV"), chemically coupled to Q β as described below.

A solution of 2mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 (5 ml) was reacted with 507. mu.l of an aqueous solution of 13mg/ml Sulfo-MBS (Pierce) for 30 minutes at 25 ℃ on a shaker. The reaction solution was then dialyzed twice against 2L of 20mM Hepes, 150mM NaCl pH7.4 for 2 hours each. 665ml of the dialyzed reaction mixture were reacted with 2.8ml of each of the corresponding 100mM peptide stock solutions (in DMSO) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

Immunization of mice:

female Balb/c mice were not adjuvanted with one of 4 angiotensin peptides coupled to Q β capsid protein. 50 μ g of total protein per sample was diluted to 200ml with PBS and injected subcutaneously on day 0 and 14 (100 ml on both sides of the abdomen). Mice were bled on day 21 and their sera were analyzed by angiotensin-specific ELISA.

ELISA

All 4 angiotensin peptides were separately coupled to bovine RNAseA using the chemical cross-linker sulfo-SPDP. ELISA plates were coated with the conjugated RNAse preparation at a concentration of 10 mg/ml. The plates were blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Preimmune sera of the same mice were also tested as controls. Control ELISA experiments performed on sera of mice immunized with irrelevant peptides cross-linked to Q.beta.or other carriers showed that the antibodies detected were specific for the respective peptide. The results are shown in FIGS. 8A-8D.

FIGS. 8A, 8B, 8C, 8D show ELISA assays for "Angio I", "Angio II", "Angio III", and "Angio IV" specific IgG antibodies, respectively, in sera from mice immunized with Angio I-IV coupled to Q β capsid protein. Q beta-Angio I, Q beta-Angio II, Q beta-Angio III and Q beta-Angio IV used in the figure represent vaccines injected into mice from which sera were obtained according to the definition of angiotensin peptide described above.

Female Balb/c mice were immunized subcutaneously with 50mg of vaccine in PBS on days 0, 14. IgG antibodies against all 4 peptides (conjugated to RNAse a), i.e., "Angio I" (fig. 8A), "Angio II" (fig. 8B), "Angio III" (fig. 8C) and "Angio IV" (fig. 8D), were measured in sera of mice vaccinated with Q β -Angio I, Q β -Angio II, Q β -Angio III and Q β -Angio IV on day 21. Preimmune sera of the same mice were also analyzed as controls. The results of the indicated serum dilutions are shown as optical density at 450 nm. The mean (including standard deviation) of 3 mice is shown. All mice vaccinated produced high IgG antibody titers against all 4 peptides tested. No angiotensin-specific antibodies were detected in the control (preimmune mice).

Example 43

Coupling of angiotensin I and angiotensin II to HBcAg-149-lys-2cys-Mut (i.e.cysteine-free HBcAg)

The following angiotensin peptides were chemically synthesized: CGGDRVYIHPF ("Angio I"), CGGDRVYIHPFHL ("Angio II"), DRVYIHPFHLGGC ("Angio III"), CDRVYIHPFHL ("Angio IV"), was used to chemically couple HBcAg-149-lys-2cys-Mut (i.e., cysteine-free HBcAg).

A solution of 0.8mg/ml HBcAg-149-lys-2cys-Mut capsid protein (see example 31) in PBS pH7.4 (1.25 ml) was reacted with 93. mu.l of a 13mg/ml aqueous solution of Sulfo-MBS (Pierce) for 30 minutes at 25 ℃ on a shaker. The reaction solution was then dialyzed overnight against 2L of 20mM Hepes, 150mM NaCl pH 7.4. After buffer exchange, the reaction solution was dialyzed for another 2 hours. The dialyzed reaction mixture was reacted with 1.8. mu.l of 100mM peptide stock solution (dissolved in DMSO) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 2L of 20mM Hepes, 150mM NaCl pH7.4, the buffer was replaced and dialyzed for an additional 2 hours.

Example 44

Coupling of angiotensin I and angiotensin II to E.coli type 1 pili

The following angiotensin peptides were chemically synthesized: CGGDRVYIHPF ("Angio I"), CGGDRVYIHPFHL ("Angio II"), DRVYIHPFHLGGC ("Angio III"), CDRVYIHPFHL ("Angio IV"), were used to chemically couple to E.coli type 1 pili.

A solution of 2.5mg/ml E.coli type 1 pili in 20mM Hepes pH7.4 was reacted with 400. mu.l of 100mM Sulfo-MBS (Pierce) aqueous solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted using a PD-10 column (Amersham-Pharmacia Biotech). The protein-containing fractions eluted from the column (containing about 1mg of protein, i.e.the derivatized fimbriae) were pooled and reacted with a 3-fold molar excess of peptide. For example, to about 500. mu.l of an eluate containing 1mg of the derivative pilus, 2.34. mu.l of a 100mM peptide stock solution (in DMSO) is added. The mixture was incubated on a shaker at 25 ℃ for 4 hours and subsequently dialyzed overnight at 4 ℃ against 2L of 20mM Hepes, 150mM NaCl pH 7.2.

Example 45

Coupling of Der pI peptide and Q beta and coupling of Der pI peptide and Q beta capsid protein of mice immunized by Q beta-Der pI vaccine

The following peptides derived from mite allergen Der pI of house dust were chemically synthesized:

CGNQSLDLAEQELVDCASQHGCH ("Der pI p 52"; amino acids 55-72, additionally containing a cysteine-glycine linker at the N-terminus), CQIYPPNANKIREALAQTHSA ("Der pI p 117"; amino acids 117-137). These peptides were chemically coupled to Q β as described below.

1ml of a solution of 2mg/mlQ β -capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 102. mu.l of an aqueous solution of 13mg/ml Sulfo-MBS (Pierce) for 30 minutes at 25 ℃ on a shaker. The reaction solution was then dialyzed twice against 2L of 20mM Hepes, 150mM NaClpH7.4 at 4 ℃ for 2 hours each. Mu.l of the dialyzed reaction mixture were reacted with 1.9. mu.l of 100mM peptide stock solution (in DMSO) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

Immunization of mice:

female Balb/c mice were inoculated with one of 2 DerpI peptides coupled to Q β capsid protein without adjuvant. Two mice were used for each vaccine. 30 μ g of total protein per sample was diluted to 200 μ l with PBS and injected subcutaneously on days 0 and 14. Mice were bled retroorbitally on day 21 and their sera were analyzed by Der pI peptide-specific ELISA.

ELISA

The Der pI peptides "Der pI p 52" and "Der pI p 117" were coupled to bovine RNAse A using the chemical cross-linker sulfo-SPDP, respectively. ELISA plates were coated with the conjugated RNAse preparation at a concentration of 10 mg/ml. The plates were blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Preimmune sera of the same mice were also tested as controls. Control ELISA experiments performed on sera of mice immunized with irrelevant peptides cross-linked to Q.beta.or other carriers showed that the antibodies detected were specific for the respective peptide. The results are shown in fig. 9A and 9B.

Fig. 9A and 9B show ELISA analysis of "Der pI p 52" (fig. 9A) and "Der pI p 117" (fig. 9B) specific IgG antibodies in sera of mice immunized with Der pI peptide coupled to Q β capsid protein. "p 52" and "p 117" used in fig. 9A and 9B represent vaccines injected to mice from which serum was isolated.

Preimmune sera of the same mice (day 0) were analyzed as controls. The results of the indicated serum dilutions are shown as optical density at 450 nm. On day 21, all vaccinated mice produced IgG antibodies specific for the vaccinated Der pI peptides, but no other antibody specific for the Der p I peptide. No antibody specific for Der pI peptide was detected before inoculation (day 0).

Both Der pI peptide vaccines were highly immunogenic in the absence of adjuvant. All vaccinated mice produced a good specific antibody response to the peptide in the vaccine formulation.

Example 46

Conjugation of Der pI peptide to HBcAg-149-lys-2cys-Mut (i.e., cysteine-free HBcAg)

The following peptides of mite allergen Der pI from house dust were chemically synthesized: der pI p52 (amino acids 55-72, additionally containing a cysteine-glycine linker at the N-terminus) CGNQSLDLAEQELVDCASQHGCH, Der pI p117 (amino acids 117-137): CQIYPPNANKIREALAQTHSA are provided. These peptides were used to chemically couple to HBcAg-149-lys-2cys-Mut (i.e., cysteine-free HBcAg).

A solution of 0.8mg/ml HBcAg-149-lys-2cys-Mut capsid protein (example 31) in PBSpH7.4 (1.25 ml) was reacted with 93. mu.l of a 13mg/ml aqueous solution of Sulfo-MBS (Pierce) on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed overnight against 2L of 20mM Hepes, 150mM NaCl pH 7.4. After buffer exchange the reaction solution was dialyzed for an additional 2 hours. The dialyzed reaction mixture was reacted with 1.8. mu.l of 100mM peptide stock solution (dissolved in DMSO) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 2L of 20mM Hepes, 150mM NaCl pH7.4, buffer exchanged, and dialyzed for an additional 2 hours.

Example 47

Coupling of Der pI peptide to Escherichia coli type 1 pilus

The following peptides from the house dust mite allergen Der pI were chemically synthesized: der pI p52 (amino acids 55-72, additionally containing a cysteine-glycine linker at the N-terminus) CGNQSLDLAEQELVDCASQHGCH, Der pI p117 (amino acids 117-137): CQIYPPNANKIREALAQTHSA are provided. These peptides were used to chemically couple to E.coli type 1 pili.

Mu.l of a 2.5mg/ml solution of E.coli type 1 pili in 20mM Hepes pH7.4 was reacted with 60. mu.l of an aqueous 100mM Sulfo-MBS (Pierce) solution on a shaker at room temperature for 60 minutes, and the reaction mixture was desalted using a PD-10 column (Amersham-Pharmacia Biotech). The protein-containing fractions eluted from the column (containing about 1mg of protein, i.e.the derivatized fimbriae) were pooled and reacted with a 3-fold molar excess of peptide. For example, to 500. mu.l of an eluate containing about 1mg of the derivative pilus, 2.34. mu.l of a 100mM peptide stock solution (in DMSO) is added. The mixture was incubated on a shaker at 25 ℃ for 4 hours and subsequently dialyzed overnight at 4 ℃ against 2L of 20mM Hepes, 150mM NaCl pH 7.2.

Example 48

Coupling of human VEGFR-II peptides to E.coli type 1 pili and immunization of mice with vaccines comprising E.coli type 1 pili-human VEGFR-II peptide arrays

Coupling of human VEGFR-II peptides to E.coli type 1 pili

The human VEGFR II peptide having the sequence CTARTELNVGIDFNWEYPSSKHQHKK was chemically synthesized for chemical coupling to e.coli type 1 pili.

A solution of 1mg/ml pilin 20mM Hepes pH7.4 in 1400. mu.l was reacted with 85. mu.l of 100mM aqueous Sulfo-MBS (Pierce) solution on a shaker at room temperature for 60 minutes. The reaction mixture was desalted using a PD-10 column (Amersham-Pharmacia Biotech). The protein-containing fractions eluted from the column (approximately 1.4mg protein) were pooled and reacted with a 2.5-fold molar excess (final volume) of human VEGFR-II peptide. For example, to 200. mu.l of an eluate containing about 0.2mg of the derivatized pilus, 2.4. mu.l of a 10mM peptide solution (dissolved in DMSO) is added. The mixture was incubated on a shaker at 25 ℃ for 4 hours and then dialyzed overnight at 4 ℃ against 2L of 20mM Hepes pH 7.2.

Immunization of mice:

female C3H-HeJ (Toll-like receptor 4 deficient, LPS non-responsive mice) and C3H-HeN (wild type) mice were inoculated with unadjuvanted human VEGFR-II peptide coupled to type 1 pilin. Approximately 100. mu.g of total protein from each sample was diluted to 200. mu.l with PBS and injected subcutaneously on days 0, 14, and 28. Mice were bled retroorbitally on days 14, 28, and 42 and analyzed by human VEGFR-II specific ELISA.

ELISA

Sera from immunized mice were assayed by ELISA using immobilized human VEGFR-II peptide and human VEGFR-II ectodomain (R & D systems GmbH, Wiesbaden).

The human VEGFR-II peptide was coupled to bovine RNAse A using the chemical cross-linker sulfo-SPDP. ELISA plates were coated with conjugated RNAse A at a concentration of 10. mu.g/ml. The extracellular domain of human VEGFR-II was adsorbed onto the plate at a concentration of 2. mu.g/ml. The plates were blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Preimmune sera of the same mice were also tested as controls. Control ELISA experiments on sera from mice immunized with the unconjugated vector showed that the antibodies detected were specific for the respective peptide. The results for human VEGFR-II peptides coupled to type 1 pili are shown in FIG. 10. Specifically, FIGS. 10A and 10B show ELISA assays for human VEGFR-II peptide and human VEGFR-II peptide ectodomain-specific IgG antibodies in mouse sera immunized with human VEGFR-II peptide and human VEGFR-II peptide ectodomain coupled to type 1 pilin, respectively.

Female C3H-HeJ (Toll-like receptor 4 deficient, LPS non-responsive) and C3H-HeN (wild type) mice were inoculated subcutaneously with 100 μ g of vaccine in PBS on days 0, 14, 28. Serum IgG antibodies were measured against the extracellular domain of human VEGFR-II peptide (coupled to RNAse A) and human VEGFR-II peptide on day 42. Preimmune sera of the same mice were also analyzed as controls. The results of the indicated serum dilutions are shown as optical density at 450 nm. The mean of 3 mice are shown (including standard deviation). All mice vaccinated gave high IgG antibody titers against the human VEGFR-II peptide and the extracellular domain (KDR) of the human VEGFR-II peptide, with no difference observed between mice lacking Toll-like receptor 4 and wild-type mice. This is noteworthy because it has been demonstrated that the formation of high IgG antibody titers against the extracellular domains of the human VEGFR-II peptide and human VEGFR-II peptide is not associated with endotoxin contamination.

Example 49

Coupling of human VEGFR-II peptides to Q β capsid proteins and immunization of mice with vaccines comprising Q β capsid protein-human VEGFR-II peptide arrays

Coupling of human VEGFR-II peptides to Q β capsid proteins

The human VEGFR-II peptide having the sequence CTARTELNVGIDFNWEYPSSKHQHKK was chemically synthesized for chemical coupling to the Q β capsid protein.

1ml of a solution of 1mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 20. mu.l of 100mM Sulfo-MBS (Pierce) in water on a shaker at room temperature for 45 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes pH7.4 twice for 2 hours at 4 ℃. Mu.l of the dialyzed reaction mixture was reacted with 12. mu.l of 10mM human VEGFR-II peptide solution (dissolved in DMSO) on a shaker at 25 ℃ for 4 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes pH7.4 at 4 ℃ for 2 times for 2 hours each.

1ml of a solution of 2mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 102. mu.l of an aqueous solution of 13mg/ml Sulfo-MBS (Pierce) for 30 minutes at 25 ℃ on a shaker. The reaction solution was then dialyzed twice against 2L of 20mM Hepes, 150mM NaClpH7.4 at 4 ℃ for 2 hours each. Mu.l of the dialyzed reaction mixture were reacted with 1.9. mu.l of 100mM peptide stock solution (in DMSO) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

Immunization of mice:

c57BL/6 mice were inoculated with human VEGFR-II peptide coupled to Q β protein without adjuvant. Approximately 50. mu.g of total protein from each sample was diluted to 200. mu.l with PBS and injected subcutaneously on days 0, 14, and 28. Mice were bled retroorbitally on days 14, 28, and 42 and analyzed by human VEGFR-II specific ELISA.

Example 50

Conjugation of human VEGFR-II peptides to HBcAg-149-lys-2cys-Mut capsid protein (i.e., cysteine-free HBcAg), and immunization of mice with vaccines comprising HBcAg-149-lys-2cys-Mut capsid protein-human VEGFR-II peptide arrays

Coupling of human VEGFR-II peptide to HBcAg-149-lys-2cys-Mut capsid protein

A human VEGFR-II peptide having the sequence CTARTELNVGIDFNWEYPSSKHQHKK was chemically synthesized for chemical coupling to the HBcAg-149-lys-2cys-Mut capsid protein.

A solution of 0.9mg/ml cysteine-free HbcAg capsid protein (see example 31) in PBSpH7.4 (3 ml) was reacted with 37.5. mu.l of 100mM Sulfo-MBS (Pierce) in water for 45 minutes at room temperature on a shaker. The reaction solution was then dialyzed overnight against 2L of 20mM Hepes pH 7.4. After buffer exchange the reaction solution was dialyzed for an additional 2 hours. The dialyzed reaction mixture was reacted with 3. mu.l of 10mM human VEGFR-II peptide solution (dissolved in DMSO) on a shaker at 25 ℃ for 4 hours. The reaction mixture was then dialyzed overnight at 4 ℃ against 2L of 20mM Hepes pH7.4, buffer exchanged, and dialyzed for an additional 2 hours.

Example 51

Construction of HBcAg1-183Lys

Hepatitis core antigen (HBcAg)1-183 was modified as described in example 23. A part of the c/e1 epitope (amino acid residues 72-88) (proline 79 and alanine 80) region was genetically engineered to be replaced by the peptide Gly-Gly-Lys-Gly-Gly (HBcAg1-183 Lys construct). The introduced lysine residue contains a reactive amino group on its side chain, which can be used for intermolecular chemical crosslinking of HBcAg particles with any antigen containing a free cysteine group. The PCR method was essentially as described in example 1, and the HBcAg1-183Lys gene was prepared by a conventional cloning technique.

The assembly into a full-length gene was performed by amplifying two different fragments of the HBcAg gene from pEco63, inserting the Gly-Lys-Gly sequence, and then fusing the two fragments by PCR, as described in example 23. The following PCR primer combinations were used:

fragment 1:

primer 1: EcoRiHBcAg(s) (see example 23)

Primer 2: Lys-HBcAg (as) (see example 23)

Fragment 2:

primer 3: Lys-HBcAg(s) (see example 23)

Primer 4: HBcAgwtwHindIII

CGCGTCCCAAGCTTCTAACATTGAGATTCCCGAGATTG

Assembling:

primer 1: EcoRiHBcAg(s) (see example 23)

Primer 2: HBcAgwtwHindIII

The assembled full-length gene was digested with EcoRI (GAATTC) and HindIII (AAGCTT) enzymes and cloned into pKK vector (Pharmacia) digested at the same restriction sites.

Example 52

Coupling of muttnf α peptides to HBcAg1-183Lys, and immunization of mice with vaccines comprising HBcAg1-183 Lys-muttnf α peptide arrays

Coupling of MuTNF alpha peptide to HBcAg1-183Lys

HBcAg1-183Lys at a concentration of 0.6mg/ml (29. mu.M) was treated with iodoacetamide as described in example 32. HBcAg1-183Lys was then reacted with a 5-fold excess of the crosslinker Sulfo-MBS as described in example 32 and dialyzed overnight at 4 ℃ against 20mM Hepes pH 7.2. Activated (derivatized) HBcAg1-183Lys was reacted with a 5-fold molar excess of the peptide mutNa (SEQ ID NO: CGGVEEQLEWLSQR, 100mM MHBcAG1-183Lys stock solution directly diluted in DMSO) at room temperature for 4 hours. The coupling reaction solution (ca.1 ml solution) was dialyzed against 2L of 20mM Hepes pH7.2 at 4 ℃ for 4 hours each. The dialyzed coupling reaction solution was equally divided into liquid nitrogen and frozen, and stored at-80 ℃ until it was used for immunization of mice.

Immunization:

two mice (female Balb/c) were immunized intravenously on day 0 and day 14 without adjuvant, 100. mu.g of HBcAg1-183Lys coupled to mutNalpha peptide per animal. Serum MuTNF α peptide (coated as ribonuclease A complex) and antibodies specific for native TNF α protein (Sigma) were assayed by ELISA on day 21.

ELISA

Murine TNF α protein (Sigma) was coated at a concentration of 2 μ g/ml. Preimmune sera of the same mice were tested as controls. FIG. 14 shows the results of ELISA experiments demonstrating that immunization with HBcAg1-183Lys (full length HBc-TNF) conjugated to mutNalpha peptide generates an immune response specific for murine TNF alpha protein. Mouse sera collected on day 0 (pre-immunization) and day 21 were tested at 3 different dilutions. Each bar is the average of the serum signals of two mice. Because the amino acid sequence of the muttnf α peptide is derived from the sequence of the mouse TNF α protein, vaccination with HBcAg1-183Lys coupled to the muttnf α peptide induces an immune response against the autoantigen.

Example 53

Coupling of 3 'TNF II peptides to 2cysLys-mut HBcAg1-149 and immunization of mice with a vaccine comprising 2cysLys-mut HBcAg 1-149-3' TNF II peptide array

Coupling of 3' TNF II peptide to 2cysLys-mut HBcAg1-149

2cysLys-mut HBcAg1-149 at a concentration of 2mg/ml was reacted with a 50-fold excess of cross-linker in 20mM Hepes, 150mM NaCl pH7.2 for 30 min at room temperature. Excess crosslinker was removed by dialysis overnight and the activated (derivatized) 2cysLys-mut HBcAg1-149 capsid protein was reacted with a 10-fold excess of 3' TNF II peptide (SEQ: SSQNSSDKPVAHVVANHGVGGC, diluted from a 100mM stock in DMSO) at room temperature for 4 hours. The reaction mixture was then dialyzed overnight in a 50000Da molecular weight dialysis tube, frozen in liquid nitrogen and stored at-80 ℃ until used to immunize mice.

Immunization of mice:

3 8-week-old female C3H/HeN mice were inoculated with 3' TNF II peptide coupled to 2cysLys-mutHBcAg1-149 without adjuvant. Mu.g of total protein was diluted to 200. mu.l with PBS and injected subcutaneously on day 0 and 14 (100. mu.l on both sides of the abdomen). Mice were bled retroorbitally on day 0 and day 21 and their sera were analyzed by ELISA specific for murine TNF α protein.

ELISA

Murine TNF α protein (Sigma) was coated at a concentration of 2 μ g/ml. Preimmune sera of the same mice were tested as controls. FIG. 15 shows the results of an ELISA experiment demonstrating that immunization with 2cysLys-mut HBcAg1-149 conjugated to a 3' TNF II peptide produced an immune response specific for the murine TNF α protein. Mouse sera collected on day 0 (pre-immunization) and day 21 were tested at 3 different dilutions. Each bar is the mean of the serum signals of 3 mice. Since the amino acid sequence of the 3 'TNF II peptide is derived from the sequence of the murine TNF α protein, vaccination with 2cysLys-mut HBcAg1-149 coupled to the 3' TNF II peptide induces an autoantigen-specific immune response.

Example 54

Coupling of Abeta 1-15, Abeta 1-27 and Abeta 33-42 peptides to Q beta, and immunization of mice with vaccines comprising an array of Q beta-Abeta peptides

A. Coupling of Ass 1-15 and Ass 33-42 peptides to the Q beta capsid protein Using Cross-linker SMPH

The following a β peptides were chemically synthesized: DAEFRHDSGYEVHHQGGC (abbreviated as "a β 1-15"), which peptide comprises the amino acid sequence of residues 1-15 of human a β fused at its C-terminus to the sequence GGC for coupling to Q β capsid proteins; and CGHGNKSGLMVGGVVIA (abbreviated as "A.beta.33-42"), which peptide comprises the amino acid sequence of residues 33-42 of human A.beta.and is fused at its N-terminus to the sequence CGHGNKS for coupling to the Q.beta.capsid protein. Both peptides were chemically coupled to Q β capsid protein as described below.

A solution of 2mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 (1.5 ml) was reacted with 16.6. mu.l of 65mM SMPH (Pierce) in water at 25 ℃ for 30 minutes on a shaker. The reaction solution was dialyzed 2 times against 2L of 20mM Hepes, 150mM NaCl pH7.4 for 2 hours at 4 ℃ in a dialysis tube with a molecular weight resistance of 10000 Da. Mu.l of the dialysis reaction mixture containing activated (derivatized) Q.beta.was reacted with 6.5. mu.l of 50mM each of the corresponding peptide stock solutions (in DMSO) on a shaker at 15 ℃ for 2 hours. Mu.l of the reaction mixture was then dialyzed overnight against 2L of 20mM Hepes, 150mM NaCl pH7.4, and the next morning the buffer was changed for an additional 2 hours. The reaction mixture was then aliquoted into liquid nitrogen and frozen and stored at-80 ℃ until used to immunize mice.

The results of the coupling experiments were analyzed by SDS-PAGE and are shown in FIG. 13A and FIG. 13B. The arrows indicate bands corresponding to one Q β subunit coupled to two peptides (fig. 13A) or one Q β subunit coupled to one peptide (fig. 13B). The molecular weights of the standard proteins are shown in the left blank of FIGS. 13A and 13B.

The samples loaded onto the gel of fig. 13A are as follows:

1: derivatized Q β; 2: q β coupled to "a β 1-15", the supernatant of the sample collected and centrifuged at the end of the coupling reaction; 3: q β coupled to "a β 1-15", a pellet of the sample collected and centrifuged at the end of the coupling reaction; 4: q β coupled to "a β 1-15", the supernatant of the undialyzed but centrifuged sample, left to stand at 4 ℃ for 24 hours; 5: q β coupled to "a β 1-15", a precipitate of an undialyzed but centrifuged sample left standing at 4 ℃ for 24 hours; 6: q β coupled to "a β 1-15", supernatant of sample collected and centrifuged after dialysis of the coupling reaction solution.

The samples loaded onto the gel of fig. 13B are as follows:

1: derivatized Q β; 2: q β coupled to "a β 33-42", the supernatant of the sample taken and centrifuged at the end of the coupling reaction; 3: q β coupled to "a β 33-42", a pellet of the sample taken and centrifuged at the end of the coupling reaction; 4: q β coupled to "a β 33-42", the supernatant of the undialyzed but centrifuged sample, left to stand at 4 ℃ for 24 hours; 5: q β coupled to "a β 33-42", a precipitate of an undialyzed but centrifuged sample left standing at 4 ℃ for 24 hours; 6: q β coupled to "a β 33-42", supernatant of sample collected and centrifuged after dialysis of the coupling reaction liquid.

B. Coupling of "Abeta 1-27" peptides to Q beta capsid protein Using Cross-linker SMPH

The following a β peptides ("a β 1-27") were chemically synthesized: DAEFRHDSGYEVHHQKLVFFAEDVGSNGGC are provided. The peptide comprises the amino acid sequence of residues 1-27 of human a β, fused at its C-terminus to the sequence GGC for coupling to Q β capsid proteins.

The first batch of "a β 1-27" coupled to Q β capsid protein, hereinafter abbreviated as "Q β -a β 1-27 batch 1", was prepared as follows:

a solution of 2mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 (1.5 ml) was reacted with 16.6. mu.l of 65mM SMPH (Pierce) in water at 25 ℃ for 30 minutes on a shaker. The reaction solution was then dialyzed 2 times against 2L of 20mM Hepes, 150mM NaCl pH7.4 in a dialysis tube with a molecular weight resistance of 10000Da at 4 ℃ for 2 hours each. Mu.l of the dialyzed reaction mixture was then reacted with 6.5. mu.l of 50mM peptide stock solution (dissolved in DMSO) on a shaker at 15 ℃ for 2 hours. 200 μ l samples were aliquoted, frozen in liquid nitrogen, and stored at-80 ℃ until used to immunize mice.

The second batch of "Α β 1-27" coupled to Q β capsid protein, hereinafter abbreviated as "Q β - Α β 1-27 batch 2", was prepared as follows:

a500. mu.l solution of Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 11.3. mu.l of 32.5mM aqueous SMPH (Pierce) solution on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed 2 times at 4 ℃ for 2 hours each against 2L of 20mM Hepes, 150mM NaCl pH7.4 in a dialysis tube (SnakeSkirn, Pierce) having a molecular weight exclusion of 3500 Da. The dialyzed reaction mixture was then reacted with 3.6. mu.l of a 50mM peptide stock solution (dissolved in DMSO) on a shaker at 15 ℃ for 2 hours. The reaction mixture was dialyzed against 1L of 20mM Hepes, 150mM NaCl pH7.4 for 1 hour, and after the last buffer change, against a 50000Da resistant dialysis membrane (Spectrpor, Spectrum) overnight. Then the reaction mixture was aliquoted into liquid nitrogen to freeze, and stored at-80 ℃ until it was used to immunize mice. "batch 1 of Q β -A β 1-27" was used for the first immunization, while "batch 2 of Q β -A β 1-27" was used for the boost.

The results of the coupling experiments were analyzed by SDS-PAGE and are shown in FIG. 13C. The arrow indicates the band corresponding to one Q β subunit coupled to one peptide.

The samples loaded onto the gel of fig. 13C are as follows:

m: and (4) protein standard. 1: q β capsid protein. 2: derivatised Q β, supernatant of samples taken at the end of the derivatisation reaction and centrifuged. 3: derivatized Q β, pellet of sample collected and centrifuged at the end of the derivatization reaction. 4: q β coupled to "a β 1-27", the supernatant of the sample taken and centrifuged at the end of the coupling reaction. 5: q β coupled to "a β 1-27", a pellet of a sample taken and centrifuged at the end of the coupling reaction. 6: q β coupled to "a β 1-27", supernatant of sample collected and centrifuged after dialysis of the coupling reaction solution. 7: q.beta.coupled to "A.beta.1-27", precipitation of a sample collected and centrifuged after dialysis of the coupled reaction solution.

C. Coupling of "Abeta 1-15" peptides to Q beta capsid protein Using the crosslinker Sulfo-GMBS

Mu.l of a solution of 2mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 5.5. mu.l of a 65mM aqueous SMPH (Pierce) solution at 25 ℃ for 30 minutes on a shaker. The reaction solution was dialyzed 2 times against 2L of 20mM Hepes, 150mM NaCl pH7.4 for 2 hours at 4 ℃ in a dialysis tube with a molecular weight resistance of 10000 Da. Then 500. mu.l of the dialyzed reaction mixture was reacted with 6.5. mu.l of 50mM peptide stock solution (dissolved in DMSO) on a shaker at 15 ℃ for 2 hours. Mu.l of the reaction mixture was then dialyzed overnight at 4 ℃ against 2L of 20mM Hepes, 150mM NaCl pH7.4, and the next morning dialysis was continued for 2 hours with buffer exchange. The reaction mixture was then aliquoted and frozen in liquid nitrogen and stored at-80 ℃ until used to immunize mice.

The results of the coupling experiments were analyzed by SDS-PAGE and are shown in FIG. 13D. Arrows indicate bands corresponding to one, two and three peptides coupled to one Q β subunit, respectively.

The samples loaded onto the gel of fig. 13D are as follows:

m: and (4) protein standard. 1: derivatized Q β. 2: q β coupled to "a β 1-15", the supernatant of the sample taken and centrifuged at the end of the coupling reaction. 3: q.beta.coupled to "A.beta.1-15", a pellet of the sample taken and centrifuged at the end of the coupling reaction. 4: q.beta.coupled to "A.beta.1-15", the supernatant of the undialyzed but centrifuged sample was left to stand at 4 ℃ for 24 hours. 5: q.beta.coupled to "A.beta.1-15", and left to stand at 4 ℃ for 24 hours, precipitation of a sample which had not been dialyzed but centrifuged. 6: q β coupled to "a β 1-15", supernatant of sample collected and centrifuged after dialysis of the coupling reaction solution.

D. Coupling of 'Abeta 1-15' and Q beta capsid protein by using crosslinker Sulfo-MBS

A solution of 500. mu. l Q β capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 14.7. mu.l of 100mM aqueous Sulfo-MBS (Pierce) solution on a shaker at 25 ℃ for 30 minutes. The reaction solution was dialyzed 2 times at 4 ℃ for 2 hours each against 2L of 20mM Hepes, 150mM NaCl pH7.4 in a dialysis tube (Snakeskin, Pierce) having a molecular weight exclusion of 3500 Da. The dialyzed reaction mixture was then reacted with 7.2. mu.l of a 50mM peptide stock solution (dissolved in DMSO) on a shaker at 15 ℃ for 2 hours. The reaction mixture was then dialyzed against 50000Da resistant dialysis membrane (Spectrpor, spectrum) for 3 times 4 hours against 2L of 20mM Hepes, 10mM NaCl pH 7.4. The reaction mixture was then aliquoted and frozen in liquid nitrogen and stored at-80 ℃ until used to immunize mice.

The results of the coupling experiments were analyzed by SDS-PAGE and are shown in FIG. 13E. The arrow indicates the band corresponding to one peptide coupled to one Q β subunit.

The samples loaded onto the gel of fig. 13E are as follows:

1: q β capsid protein. 2: derivatised Q β, supernatant of samples taken at the end of the derivatisation reaction and centrifuged. 3: derivatized Q β, pellet of sample collected and centrifuged at the end of the derivatization reaction. 4: derivatized Q β, supernatant of a sample collected and centrifuged at the end of the derivatization reaction solution dialysis. 5: derivatized Q β, pellet of sample collected and centrifuged at the end of dialysis of the derivatized reaction. 6: q β coupled to "a β 1-15", the supernatant of the sample taken and centrifuged at the end of the coupling reaction. 7: q.beta.coupled to "A.beta.1-15", a pellet of the sample taken and centrifuged at the end of the coupling reaction. 8: q β coupled to "a β 1-15", supernatant of sample collected and centrifuged after dialysis of the coupling reaction solution.

E. Immunization of mice

5 groups of 8 week old female C57BL/6 mice, 3 per group, were inoculated with one of 5A β peptide-Q β capsid protein conjugates without adjuvant. 25 μ g of total protein per sample was diluted to 200 μ l with PBS and injected subcutaneously on days 0 and 14. Mice were bled retroorbitally on day 0 (pre-immunization) and day 21 and their sera were analyzed by ELISA. The "A β 1-15" peptide was coupled to Q β using 3 different cross-linking agents, resulting in 3 different vaccine formulations ("Q β -A β 1-15 SMPH", "Q β -A β 1-15 SMBS", "Q β -A β 1-15 SGMBS", see ELISA section for results).

F.ELISA

All 3 a β peptides were separately coupled to bovine RNAseA using the chemical cross-linker SPDP as follows: a solution of 10mg of RNAse in 2mL PBS (50mM phosphate buffer, 150mM NaCl, pH7.2) was reacted with 100. mu.l of a 20mM SPDP solution in DMSO on a shaker at 25 ℃ for 60 minutes. The activated (derivatised) RNAse was separated from the excess cross-linking agent by gel filtration using a PD10 column (Pharmacia). The protein-containing fractions were combined and concentrated to a volume of 2ml using a centrifugal filter (5000 MWCO). 333 μ l of derivatized RNAseA solution was reacted with 2 μ l of peptide stock solution (50mM in DMSO). The coupling reaction was detected spectrophotometrically.

ELISA plates were coated with RNAse A coupled to the peptide at a concentration of 10. mu.g/ml. The plates were blocked and then incubated with serial dilutions of mouse serum. Bound antibody was detected with an enzyme-labeled anti-mouse IgG antibody. Preimmune sera or control sera of mice immunized with irrelevant peptides coupled to Q β showed that the detected antibodies were specific for the respective peptide. FIGS. 14A, 14B and 14C show ELISA assays for "A β 1-15", "A β 1-27" and "A β 33-42" specific IgG antibodies in sera of mice immunized with "A β 1-15", "A β 1-27" and "A β 33-42", respectively, conjugated to Q β capsid protein. The units on the abscissa represent the vaccines injected into serum-collected mice, and the peptides and cross-linking agents used to prepare each vaccine are described. All sera were assayed with three peptides coupled to RNAseA and the results showed that there was cross-reactivity between anti-peptides 1-15 and anti-1-27 antibodies, which was not observed with anti-33-42 antibodies, demonstrating the specificity of the immune response. Also, the ELISA titers obtained (expressed as serum dilutions that produced ELISA signals 3 standard deviations above background) were very high, 60000-600000. No antibody specific for a β peptide was detected in the control (preimmune mice).

Example 55

Introduction of cysteine-containing linkers, expression and purification of anti-idiotype IgE mimobodies, and coupling to Q beta-capsid proteins

Construction of mimobody expression plasmids for coupling with Q β capsid proteins

The plasmid is based on the expression plasmid VAE051-pASK 116. The plasmid contains coding regions for the heavy and light chains of the mimobody. To introduce a cysteine-containing linker at the C-terminus of the heavy chain, the following primers were used:

primer CA 2F:

CGGCTCGAGCATCACCATCACCATCACGGTGAAGTTAAACTGCAGCTGGAGTCG

primer CA 1R:

CATGCCATGGTTAACCACAGGTGTGGGTTTTATCACAAGATTTGGGCTCAAC

primer CB 1R:

CATGCCATGGTTAACCACACGGCGGAGAGGTGTGGGTTTTATCACAAGATTTGGGCTCAAC

primer CC 1R:

CCAGAAGAACCCGGCGGGGTAGACGGTTTCGGGCTAGCACAAGATTTGGGCTCAACTC

primer CC 1F:

CGCCGGGTTCTTCTGGTGGTGCTCCGGGTGGTTGCGGTTAACCATGGAGAAAATAAAGTG

primer CCR 2:

CTCCCGGGTAGAAGTCAC

construction of pCA2

A741 bp fragment encoding the heavy chain portion containing an extended sequence encoding a cysteine-containing linker sequence was amplified with primers CA2F and CA 1R. VAE-pASK116 was used as a template for Pfx polymerase (Roche) and denaturation was started at 92 ℃ using a PCR instrument (Robo), cycle: 30s at 92 ℃, 30s at 48 ℃, 60s at 68 ℃ for 5 cycles, and then 30 cycles at 92 ℃ for 30s, 58 ℃ for 30s, and 1min at 68 ℃. The appropriately sized PCR product was purified using the Qiagen PCR purification kit and digested with XhoI and NcoI according to the manufacturer's recommendations (Gibco). The product was purified from agarose gel using Qiagen gel extraction kit. In parallel, plasmid VAE-pASK116 was digested with XhoI and NcoI and the 3.7kb band was purified from an agarose gel. An appropriate aliquot of XhoI-NcoI digested PCR product was ligated with the plasmid overnight at 16 ℃ using T4DNA ligase according to the manufacturer's instructions (Gibco). Competent E.coli XL-1 cells were transformed with the ligation products and plated on chloramphenicol-containing agarose plates. A single colony was amplified in LB/chloramphenicol medium, a plasmid (Qiagen mini plasmid kit) was prepared, and the presence of the XhoI-NcoI insert was detected after digestion with the appropriate enzymes. The corresponding positive plasmid was designated pCA2 and both strands were sequenced to confirm the identity of the plasmid containing the cysteine linker.

Construction of pCB2

Linker 2 was introduced at the 5' end of the heavy chain coding sequence using primers CA2F and CB1R under the same conditions as described in section a.1. The resulting PCR product was 750bp and cloned into VAE051-pASK116 as described in section A.1.

Construction of pCC2

Plasmid pCC2 was constructed in two steps: a first PCR product of 754bp was amplified using primers CA2F and CC 1R. A second PCR product of 560bp was generated with primers CC1F and CC 2R. Both PCRs were performed using VAE051-pASK116 as template, as described in section A.1. Both PCR products were separated from agarose gel, mixed with primers CA2F and CC2R and subjected to a third PCR, yielding a 1298bp fragment. The fragment was isolated and digested with XhoI and NcoI. The resulting 780bp fragment was cloned into VAE-pASK100 as described in section A.1.

Expression of mimobodies

Competent E.coli W3110 cells were transformed with plasmids pCA2, pCB2 and pCC 2. A single colony from a chloramphenicol agarose plate was amplified overnight at 37 ℃ in liquid medium (LB + 15. mu.g/ml chloramphenicol). The overnight cultures were then inoculated with 1: 50v/v 1LTB medium and grown to OD600 ═ 3 at 28 ℃. 1mg/l of anhydrous tetracycline is used to induce expression. Cells were harvested after overnight culture and centrifugation at 6000 rpm. The cell pellet was incubated in lysis buffer supplemented with polymyxin B sulfate at 4 ℃ for 2 hours, from which periplasm was isolated. The spheroblasts were separated by centrifugation at 6000 rpm. The supernatant obtained contains mimobody and is dialyzed against 20mM Tris, pH 8.0.

Purification of mimobodies

The introduced his 6-tail allowed mimobody pCA2 and pCB2 to be purified by Ni-NTA fast flow (Qiagen) chromatography according to the manufacturer's recommendations. The repurification step was performed on a protein G fast flow column (Amersham Pharmacia Biotech) as necessary. Mimobody was eluted with 0.1M glycine pH2.7, immediately neutralized with NaOH and dialyzed against 20mM Hepes, 150mM NaCl, pH 7.2.

pCC2 was purified only by protein G affinity chromatography. Purity was analyzed by SDS-PAGE.

The protein sequence of the Mimobody is translated from the cDNA sequence. The N-terminal sequence was confirmed by Edman sequencing of pCA2 and pCB 2.

The light chain sequences of pCA2, pCB2, and pCC2 are identical as follows:

DIELVVTQPASVSGSPGQSITISCTGTRSDVGGYNYVSWYQQHPGKAPKL

MIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTL

GVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT

VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQ

VTHEGSTVEKTVAPTECS

pCA2 heavy chain sequence:

EVKLQLEHHHHHHGEVKLQLESGPGLVKPSETLSLTCTVSGGSISSGGYY

WTWIRQRPGKGLEWIGYTYYSGSTSYNPSLKSRVTMSVDTSKNQFSLRLT

SVTAADTAVYYCARERGETGLYYPYYYIDVWGTGTTVTVSSASTKGPSV

FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC

G

pCB2 heavy chain sequence:

EVKLQLEHHHHHHGEVKLQLESGPGLVKPSETLSLTCTVSGGSISSGGYY

WTWIRQRPGKGLEWIGYIYYSGSTSYNPSLKSRVTMSVDTSKNQFSLRLT

SVTAADTAVYYCARERGETGLYYPYYYIDVWGTGTTVTVSSASTKGPSV

FPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTS

PPCG

pCC2 heavy chain sequence:

EVKLQLEHHHHHHGEVKLQLESGPGLVKPSETLSLTCTVSGGSISSGGYY

WTWIRQRPGKGLEWIGYIYYSGSTSYNPSLKSRVTMSVDTSKNQFSLRLT

SVTAADTAVYYCARERGETGLYYPYYYIDVWGTGTTVTVSSASTKGPSV

FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCASPKPS

TPPGSSGGAPGGC

coupling of mimobodies to Q beta capsid proteins

Coupling of mimobody pCC2 to Q β capsid protein

1.25ml of a solution of 4.5mg/mlQ β -capsid protein in 20mM hepes, 150mM NaCl pH7.2 was reacted with 40. mu.l of SMPH solution (Pierce) (100 mM stock solution in DMSO) on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. Then 6. mu.l of the dialyzed reaction mixture was reacted with 30. mu.l of pCC2 solution (2.88mg/ml) on a shaker at 25 ℃ overnight.

The reaction products were analyzed on a 16% SDS-PAGE gel under reducing conditions. The gel was stained with Coomassie Brilliant blue or blotted onto nitrocellulose. The membrane was blocked and incubated with polyclonal rabbit anti-Qb antiserum (1: 2000 dilution) or mouse monoclonal anti-Fab-mAb antibody (Jackson ImmunoResearch) (1: 2000 dilution). The blot was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG (1: 7000 dilution), respectively.

The results are shown in fig. 13A. The conjugate products and the educts were analyzed on a 16% SDS-PAGE gel under reducing conditions. In fig. 13A, "pCC 2" corresponds to the mimobody prior to conjugation. "Q β -derived" represents Q β derived prior to coupling and "Q β -pCC 2" represents the product of the coupling reaction. The gel was stained with Coomassie Brilliant blue or blotted onto nitrocellulose. The membrane was blocked and incubated with polyclonal rabbit anti-Qb antiserum (1: 2000 dilution) or mouse monoclonal anti-Fab-mAb (Jackson ImmunoResearch) (1: 2000 dilution). The blot was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG (1: 7000 dilution), respectively. Immunoreactive bands were visualized with enhanced chemiluminescence reagents (Amersham Pharmacia ELC kit). Molecular weights of the standard proteins are shown in the left blank.

A coupling product of about 40kDa could be detected (fig. 13A, arrow). Its reactivity with anti-Q β antisera and anti-Fab recognizing mimobodies clearly confirms the covalent coupling of mimobodies to Q β.

Coupling of mimobody pCA2 and pCB2 to Q.beta.capsid protein

1.25ml of a solution of 4.5mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 40. mu.l of SMPH solution (Pierce) from a 100mM stock solution in DMSO on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. pCA2(1.2mg/ml) was reduced with 20mM TCEP at 25 ℃ for 30 minutes and pCB2(4.2mg/ml) was reduced with 50mM mercaptoethylamine at 37 ℃. Both mimobodies were then dialyzed 2 times at 4 ℃ against 20mM Hepes, 150mM NaCl pH 7.2. Mu.l of derivatized Q.beta.was added to 30. mu.l of mimobody and coupling was performed overnight on a shaker at 25 ℃.

The reaction products were analyzed on a 16% SDS-PAGE gel under reducing conditions. The gel was stained with Coomassie Brilliant blue or blotted onto nitrocellulose. The membrane was blocked and incubated with polyclonal rabbit anti-Q.beta.antiserum (Cytos, 1: 2000 dilution) or mouse monoclonal anti-his 6-mAb antibody (Qiagen) (1: 5000 dilution). The blot was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG (1: 5000 dilution), respectively.

The results are shown in fig. 13B and 13C. The conjugate products and the educts were analyzed on a 16% SDS-PAGE gel under reducing conditions. In fig. 15A and 15B, "pCA 2" and "pCB 2" correspond to mimobodies before coupling. "Q β -derived" represents Q β derived prior to coupling, "Q β -pCA 2" and "Q β -pCB 2" represent the products of the coupling reaction. The gel was stained with Coomassie Brilliant blue or blotted onto nitrocellulose. The membrane was blocked and incubated with polyclonal rabbit anti-Qb antiserum (1: 2000 dilution) or mouse monoclonal anti-his 6-mAb antibody (Qiagen) (1: 5000 dilution). The blot was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG (1: 5000 dilution), respectively. Immunoreactive bands were visualized with enhanced chemiluminescence reagents (Amersham Pharmacia ELC kit). Molecular weights of the standard proteins are shown in the left blank.

Both pCA2 and pCB2 conjugates were able to detect a conjugation product of about 40kDa (FIG. 15A and FIG. 15B, indicated by arrows). Its reactivity with anti-Q β antisera and anti-his 6 antibodies recognizing the mimobody heavy chain clearly confirms the covalent coupling of mimobodies to Q β.

Example 56

Coupling of Flag peptides to wild-type and mutant Q β capsid proteins using the crosslinker Sulfo-GMBS

Chemically synthesized Flag peptide, to which a CGG sequence was added for coupling to the N-terminus of the peptide, having the following sequence: CGGDYKDDDDK are provided. The peptide is chemically coupled to a wild-type Q β capsid protein and a mutant Q β capsid protein, as described below.

Coupling of flag peptide to Q β capsid protein

A solution of 2mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.2, 100. mu.l was reacted with 7. mu.l of 65mM Sulfo-GMBS aqueous solution (Pierce) on a shaker at 25 ℃ for 60 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaClpH7.2 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed reaction mixture was then reacted with 0.58. mu.l of 100mM Flag peptide stock solution (dissolved in water) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

Coupling of flag peptide to Q beta-240 capsid protein

A solution of 2mg/mlQ β -240 capsid protein in 20mM Hepes, 150mM NaCl pH7.2, 100. mu.l was reacted with 7. mu.l of 65mM Sulfo-GMBS in water (Pierce) on a shaker at 25 ℃ for 60 minutes. The reaction solution was then dialyzed 2 times against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 hours each. Mu.l of the dialyzed reaction mixture was then reacted with 0.58. mu.l of 100mM Flag peptide stock solution (dissolved in water) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each.

Coupling of flag peptide to Q beta-250 capsid protein

A solution of 2mg/ml Q β -250 capsid protein in 20mM Hepes, 150mM NaCl pH7.4 (100. mu.l) was reacted with 7. mu.l of 65mM Sulfo-GMBS in water (Pierce) on a shaker at 25 ℃ for 60 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times each for 2 hours. Mu.l of the dialyzed reaction mixture was then reacted with 0.58. mu.l of 100mM Flag peptide stock solution (dissolved in water) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each.

Coupling of flag peptide to Q beta-259 capsid protein

Mu.l of a solution of 2mg/mlQ beta-259 capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 7. mu.l of 65mM aqueous Sulfo-GMBS solution (Pierce) on a shaker at 25 ℃ for 60 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed reaction mixture was then reacted with 0.58. mu.l of 100mM Flag peptide stock solution (dissolved in water) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

The results of the coupling reaction of Q β mutants 240, 250 and 259 with Flag peptide were analyzed by SDS-PAGE and are shown in fig. 22A. The sample loading mode is as follows:

1, derivatized Q β -240; 2, Q β -240 coupled to a Flag peptide; 3, derivatized Q β -250; 4, Q β -250 coupled to a Flag peptide; 5, derivatized Q β -259; 6, Q β -259 coupled to a Flag peptide; 7, a derived wild-type Q β; 8, wild-type Q β coupled to Flag peptide; 9, protein standard.

Comparison of the derivatization reaction with the coupling reaction showed that for all mutants and wild type, coupling bands corresponding to 1 and 2 peptides per subunit were visible. The band corresponding to the uncoupled Q β subunit is extremely weak, indicating that almost all of the subunits are reactive with at least one Flag peptide. For the Q β -250 mutation and the wild type Q β, a band corresponding to 3 peptides per subunit was seen. The intensity ratio of the band corresponding to 2 peptides per subunit to the band corresponding to 1 peptide per subunit was highest for the wild type, with a ratio of 1: 1. This ratio was also higher for the Q β -250 mutant, but the Q β -240 mutant was significantly weaker and the Q β -259 mutant was the weakest.

Example 57

Coupling of the Cross-linker Sulfo-MBS, flag peptide with Q beta capsid protein

Chemically synthesized Flag peptide, to which a CGG sequence was added for coupling to the N-terminus of the peptide, having the following sequence: CGGDYKDDDDK are provided. The peptide is chemically coupled to a wild-type Q β capsid protein and a mutant Q β capsid protein, as described below.

Coupling of flag peptide to Q β capsid protein

A solution of 2mg/ml Q.beta.capsid protein in 20mM Hepes, 150mM NaCl pH7.2, 100. mu.l was reacted with 7. mu.l of a 65mM aqueous solution of Sulfo-MBS (Pierce) on a shaker at 25 ℃ for 60 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed reaction mixture was then reacted with 0.58. mu.l of 100mM FLAg peptide stock solution (in water) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each.

Coupling of flag peptide to Q beta-240 capsid protein

A solution of 2mg/ml Q.beta. -240 capsid protein in 20mM Hepes, 150mM NaCl pH7.2, 100. mu.l was reacted with 7. mu.l of a 65mM aqueous solution of Sulfo-MBS (Pierce) on a shaker at 25 ℃ for 60 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed reaction mixture was then reacted with 0.58. mu.l of 100mM FLAg peptide stock solution (in water) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each.

Coupling of flag peptide to Q beta-250 capsid protein

A solution of 2mg/ml Q β -250 capsid protein in 20mM Hepes, 150mM NaCl pH7.2, 100. mu.l was reacted with 7. mu.l of a 65mM aqueous solution of Sulfo-MBS (Pierce) on a shaker at 25 ℃ for 60 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times each for 2 hours. Mu.l of the dialyzed reaction mixture was then reacted with 0.58. mu.l of 100mM Flag peptide stock solution (dissolved in water) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each.

Coupling of flag peptide to Q beta-259 capsid protein

A solution of 2mg/ml Q β -259 capsid protein in 20mM Hepes, 150mM NaCl pH7.2, 100. mu.l was reacted with 7. mu.l of a 65mM aqueous solution of Sulfo-MBS (Pierce) on a shaker at 25 ℃ for 60 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 450mM NaCl pH7.2 at 4 ℃ for 2 hours each. Mu.l of the dialyzed reaction mixture was then reacted with 0.58. mu.l of 100mM FLAg peptide stock solution (in water) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.2 at 4 ℃ for 2 times for 2 hours each.

The results of the coupling reaction of Q β mutants 240, 250 and 259 with Flag peptide were analyzed by SDS-PAGE and are shown in fig. 1. The sample loading mode is as follows:

1. protein standards; 2. derivatized Q β -240; 3. q β -240 coupled to a Flag peptide; 4. derivatized Q β -250; 5. q β -250 coupled to a Flag peptide; 6. derivatized Q β -259; 7. q β -259 coupled to a Flag peptide; 8. a derived wild-type Q β; 9. wild-type Q β coupled to Flag peptide.

Comparison of the derivatization reaction with the coupling reaction shows that for all mutant and wild types a coupling band corresponding to 1 peptide per subunit is visible. Bands corresponding to 2 peptides per subunit are also visible for mutant Q β -250 and wild-type Q β. The intensity ratio of the band corresponding to 1 peptide per subunit to the band corresponding to the uncoupled subunit was higher for the Q β -250 mutant and the wild-type Q β. For the Q β -240 mutant, a weak band corresponding to 2 peptides per subunit was seen.

Example 58

Coupling of Flag peptide to Q beta mutant Using Cross-linker SMPH

Chemically synthesized Flag peptide, to which a CGG sequence was added for coupling to the N-terminus of the peptide, having the following sequence: CGGDYKDDDDK are provided. This peptide was chemically coupled to Q β mutants as described below.

Coupling of flag peptide to Q.beta. -240 capsid protein

100. mu.l of a solution of 2mg/ml Q.beta. -240 capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 2.94. mu.l of a solution of 100mM SMPH in DMSO (Pierce) on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. 90 μ l of the dialyzed reaction mixture was then reacted with 1.3 μ l of 50mM Flag peptide stock solution (dissolved in DMSO) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

Coupling of flag peptides to Q beta-250 capsid proteins

100. mu.l of a solution of 2mg/ml Q.beta. -250 capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 2.94. mu.l of a solution of 100mM SMPH in DMSO (Pierce) on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. 90 μ l of the dialyzed reaction mixture was then reacted with 1.3 μ l of 50mM Flag peptide stock solution (dissolved in DMSO) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

Coupling of flag peptide to Q beta-259 capsid protein

100. mu.l of a solution of 2mg/ml Q.beta. -259 capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 2.94. mu.l of a solution of 100mM SMPH in DMSO (Pierce) on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. 90 μ l of the dialyzed reaction mixture was then reacted with 1.3 μ l of 50mM Flag peptide stock solution (dissolved in DMSO) on a shaker at 25 ℃ for 2 hours. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

The results of the coupling reaction of Q β mutants 240, 250 and 259 with Flag peptide were analyzed by SDS-PAGE and are shown in fig. 1. The sample loading mode is as follows: 1. protein standards; 2Q β -240 coupled with Flag, precipitation of the coupling reaction; 3. q β -240 coupled with Flag, supernatant of the coupling reaction; SMPH-derived Q β -240; 5. q β -250 coupled with Flag, precipitation of coupling reaction; 6. q β -250 coupled with Flag, supernatant of the coupling reaction; SMPH-derived Q β -250; 8. q β -259 coupled with Flag, precipitate of coupling reaction; 9. q β -259 coupled with Flag, supernatant of the coupling reaction; SMPH-derived Q β -259.

Comparison of the derivatization and coupling reactions showed that for all mutants, coupling bands corresponding to 1 and 2 peptides per subunit were visible. For the Q β -250 mutant, bands corresponding to 3 and 4 peptides per subunit were also visible.

Example 59

PLA₂Coupling of Cys protein to mutant Q.beta.capsid protein

Lyophilized mutant Q β capsid proteins were solubilized overnight in 20mM Hepes, 150mM NaCl ph 7.4.

A.PLA₂Coupling of Cys protein to Q.beta. -240 capsid protein

100. mu.l of a solution of 2mg/ml Q.beta. -240 capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 2.94. mu.l of a solution of 100mM SMPH in DMSO (Pierce) on a shaker at 25 ℃ for 30 minutes. The reaction solution was dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times each for 2 hours. 90 μ l of the dialyzed reaction mixture was then mixed with 146 μ l of 20mM Hepes, 150mM NaCl pH7.4, and 85.7 μ l of 2.1mg/ml PLA₂Cys stock solution was reacted for 4 hours at 25 ℃ on a shaker. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

B.PLA₂Coupling of Cys protein to Q beta-250 capsid protein

100. mu.l of a solution of 2mg/ml Q.beta. -250 capsid protein in 20mM Hepes, 150mM NaCl pH7.4 was reacted with 2.94. mu.l of a solution of 100mM SMPH in DMSO (Pierce) on a shaker at 25 ℃ for 30 minutes. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed reaction mixture was mixed with 146. mu.l of 20mM Hepes, 150mM NaCl pH7.4, and 85.7. mu.l of 2.1mg/ml PLA ₂Cys stock solution was reacted for 4 hours at 25 ℃ on a shaker. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

C.PLA₂Coupling of Cys protein to Q beta-259 capsid protein

100. mu.l and 2.94. mu.l of a solution of 2mg/ml Q.beta. -259 capsid protein in 20mM Hepes, 150mM NaCl pH7.4A solution of mM SMPH in DMSO (Pierce) was reacted for 30 minutes on a shaker at 25 ℃. The reaction solution was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each. Mu.l of the dialyzed reaction mixture was mixed with 146. mu.l of 20mM Hepes, 150mM NaCl pH7.4, and 85.7. mu.l of 2.1mg/ml PLA₂Cys stock solution was reacted for 4 hours at 25 ℃ on a shaker. The reaction mixture was then dialyzed against 2L of 20mM Hepes, 150mM NaCl pH7.4 at 4 ℃ for 2 times for 2 hours each.

The results of the coupling experiments analyzed by SDS-PAGE are shown in FIG. 1. The sample loading mode is as follows: 1. and (4) protein standard. 2. Derivatized Q β -240; 3. with PLA₂-Cys-coupled Q β -240, the supernatant of the coupling reaction; 4 with PLA₂-Cys-coupled Q β -240, precipitation of the coupling reaction; 5. derivatized Q β -250; 6. with PLA₂-Cys-coupled Q β -250, supernatant of the coupling reaction; 7. with PLA₂-Cys-coupled Q β -250, precipitation of the coupling reaction; 8. derivatized Q β -259; 9. with PLA ₂-Cys-coupled Q β -259, supernatant of the coupling reaction; 10. with PLA₂-Cys coupled Q β -259, precipitation of the coupling reaction; PLA 11₂-Cys。

The coupling bands (indicated by arrows in the figure) were visible for all mutants, indicating PLA₂Cys protein can be coupled to all mutant Q β capsid proteins.

All patents and publications mentioned herein are incorporated herein by reference.

The entire disclosures of U.S. application No. 09/449,631 and WO 00/3227 filed 11/30 1999 are hereby incorporated by reference in their entirety. All publications and patents mentioned above are herein incorporated by reference in their entirety.

Sequence listing

<110> Setos Biotechnology Co

Renner，Wolfgang A.

Bachmann，Martin

Tissot，Alain

Maurer，Patrick

Lechner，Franziska

Sebbel，Peter

Piossek，Christine

Ortmann，Rainer

Luond，Rainer

Staufenbiel，Matthias

Frey，Peter

<120> molecular antigen array

<130>1700.019PC07

<140>PCT/IB02/00168

<141>2002-01-21

<150>US 60/262,379

<151>2001-01-19

<150>US60/288,549

<151>2001-05-04

<150>US 60/326,998

<151>2001-10-05

<150>US 60/331,045

<151>2001-11-07

<160>350

<170>PatentIn version 3.1

<210>1

<211>41

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>1

ggggacgcgt gcagcaggta accaccgtta aagaaggcac c 41

<210>2

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>2

cggtggttac ctgctgcacg cgttgcttaa gcgacatgta gcgg 44

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>3

ccatgaggcc tacgataccc 20

<210>4

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>4

ggcactcacg gcgcgcttta caggc 25

<210>5

<211>47

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>5

ccttctttaa cggtggttac ctgctggcaa ccaacgtggt tcatgac 47

<210>6

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>6

aagcatgctg cacgcgtgtg cggtggtcgg atcgcccggc 40

<210>4

<211>90

<212>DNA

<213> Artificial sequence

<220>

<223>olginucleotide primer

<400>7

gggtctagat tcccaaccat tcccttatcc aggctttttg acaacgctat gctccgcgcc 60

catcgtctgc accagctggc ctttgacacc 90

<210>8

<211>108

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>8

gggtctagaa ggaggtaaaa aacgatgaaa aagacagctatcgcgattgc agtggcactg 60

gctggtttcg ctaccgtagc gcaggccttc ccaaccattc ccttatcc 108

<210>9

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>9

cccgaattcc tagaagccac agctgccctc c 31

<210>10

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>10

cctgcggtgg tctgaccgac accc 24

<210>11

<211>41

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>11

ccgcggaaga gccaccgcaa ccaccgtgtg ccgccaggat g 41

<210>12

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>12

ctatcatcta gaatgaatag aggattcttt aac 33

<210>13

<211>15

<212>DNA

<213> Artificial sequence

<220>

<223> modified ribosome binding site

<400>13

aggaggtaaa aaacg 15

<210>14

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> Signal peptide

<400>14

Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala

1 5 10 15

Thr Val Ala Gln Ala

20

<210>15

<211>46

<212>PRT

<213> Artificial sequence

<220>

<223> modified Fos constructs

<400>15

Cys Gly Gly Leu Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Val Glu

1 5 10 15

Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu

20 25 30

Leu Glu Lys Leu Glu Phe Ile Leu Ala Ala His Gly Gly Cys

35 40 45

<210>16

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400>16

Ala Ala Ala Ser Gly Gly

1 5

<210>17

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400>17

Gly Gly Ser Ala Ala Ala

1 5

<210>18

<211>256

<212>DNA

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>18

gaattcagga ggtaaaaaac gatgaaaaag acagctatcg cgattgcagt ggcactggct 60

ggtttcgcta ccgtagcgca ggcctgggtg ggggcggccg cttctggtgg ttgcggtggt 120

ctgaccgaca ccctgcaggc ggaaaccgac caggtggaag acgaaaaatc cgcgctgcaa 180

accgaaatcg cgaacctgct gaaagaaaaa gaaaagctgg agttcatcct ggcggcacac 240

ggtggttgct aagctt 256

<210>19

<211>52

<212>PRT

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>19

Ala Ala Ala Ser Gly Gly Cys Gly Gly Leu Thr Asp Thr Leu Gln Ala

1 5 10 15

Glu Thr Asp Gln Val Glu Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile

20 25 30

Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala

35 40 45

His Gly Gly Cys

50

<210>20

<211>261

<212>DNA

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<220>

<221>CDS

<222>(22)..(240)

<400>20

gaattcagga ggtaaaaaac g atg aaa aag aca gct atc gcg att gca gtg 51

Met Lys Lys Thr Ala Ile Ala Ile Ala Val

1 5 10

gca ctg gct ggt ttc gct acc gta gcg cag gcc tgc ggt ggt ctg acc 99

Ala Leu Ala Gly Phe Ala Thr Val Ala Gln Ala Cys Gly Gly Leu Thr

15 20 25

gac acc ctg cag gcg gaa acc gac cag gtg gaa gac gaa aaa tcc gcg 147

Asp Thr Leu Gln Ala Glu Thr Asp Gln Val Glu Asp Glu Lys Ser Ala

30 35 40

ctg caa acc gaa atc gcg aac ctg ctg aaa gaa aaa gaa aag ctg gag 195

Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu

45 50 55

ttc atc ctg gcg gca cac ggt ggt tgc ggt ggt tct gcg gcc gct 240

Phe Ile Leu Ala Ala His Gly Gly Cys Gly Gly Ser Ala Ala Ala

60 65 70

gggtgtgggg atatcaagct t 261

<210>21

<211>73

<212>PRT

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>21

Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala

1 5 10 15

Thr Val Ala Gln Ala Cys Gly Gly Leu Thr Asp Thr Leu Gln Ala Glu

20 25 30

Thr Asp Gln Val Glu Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile Ala

35 40 45

Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala His

50 55 60

Gly Gly Cys Gly Gly Ser Ala Ala Ala

65 70

<210>22

<211>196

<212>DNA

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<220>

<221>CDS

<222>(34)..(189)

<400>22

gaattcagga ggtaaaaaga tatcgggtgt ggg gcg gcc gct tct ggt ggt tgc 54

Ala Ala Ala Ser Gly Gly Cys

1 5

ggt ggt ctg acc gac acc ctg cag gcg gaa acc gac cag gtg gaa gac 102

Gly Gly Leu Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Val Glu Asp

10 15 20

gaa aaa tcc gcg ctg caa acc gaa atc gcg aac ctg ctg aaa gaa aaa 150

Glu Lys Ser Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys

25 30 35

gaa aag ctg gag ttc atc ctg gcg gca cac ggt ggt tgc taagctt 196

Glu Lys Leu Glu Phe Ile Leu Ala Ala His Gly Gly Cys

40 45 50

<210>23

<211>52

<212>PRT

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>23

Ala Ala Ala Ser Gly Gly Cys Gly Gly Leu Thr Asp Thr Leu Gln Ala

1 5 10 15

Glu Thr Asp Gln Val Glu Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile

20 25 30

Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala

35 40 45

His Gly Gly Cys

50

<210>24

<211>204

<212>DNA

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>24

gaattcagga ggtaaaaaac gatggcttgc ggtggtctga ccgacaccct gcaggcggaa 60

accgaccagg tggaagacga aaaatccgcg ctgcaaaccg aaatcgcgaa cctgctgaaa 120

gaaaaagaaa agctggagtt catcctggcg gcacacggtg gttgcggtgg ttctgcggcc 180

gctgggtgtg gggatatcaa gctt 204

<210>25

<211>56

<212>PRT

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>25

Lys Thr Met Ala Cys Gly Gly Leu Thr Asp Thr Leu Gln Ala Glu Thr

1 5 10 15

Asp Gln Val Glu Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile Ala Asn

20 25 30

Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala His Gly

35 40 45

Gly Cys Gly Gly Ser Ala Ala Ala

50 55

<210>26

<211>26

<212>PRT

<213>Homo sapiens

<400>26

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala

20 25

<210>27

<211>262

<212>DNA

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>27

gaattcaggc ctatggctac aggctcccgg acgtccctgc tcctggcttt tggcctgctc 60

tgcctgccct ggcttcaaga gggcagcgct gggtgtgggg cggccgcttc tggtggttgc 120

ggtggtctga ccgacaccct gcaggcggaa accgaccagg tggaagacga aaaatccgcg 180

ctgcaaaccg aaatcgcgaa cctgctgaaa gaaaaagaaa agctggagtt catcctggcg 240

gcacacggtg gttgctaagc tt 262

<210>28

<211>52

<212>PRT

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>28

Ala Ala Ala Ser Gly Gly Cys Gly Gly Leu Thr Asp Thr Leu Gln Ala

1 5 10 15

Glu Thr Asp Gln Val Glu Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile

20 25 30

Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala

35 40 45

His Gly Gly Cys

50

<210>29

<211>261

<212>DNA

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<220>

<221>CDS

<222>(7)..(240)

<400>29

gaattc atg gct aca ggc tcc cgg acg tcc ctg ctc ctg gct ttt ggc 48

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly

1 5 10

ctg ctc tgc ctg ccc tgg ctt caa gag ggc agc gct tgc ggt ggt ctg 96

Leu Leu Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Cys Gly Gly Leu

15 20 25 30

acc gac acc ctg cag gcg gaa acc gac cag gtg gaa gac gaa aaa tcc 144

Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Val Glu Asp Glu Lys Ser

35 40 45

gcg ctg caa acc gaa atc gcg aac ctg ctg aaa gaa aaa gaa aag ctg 192

Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu

50 55 60

gag ttc atc ctg gcg gca cac ggt ggt tgc ggt ggt tct gcg gcc gct 240

Glu Phe Ile Leu Ala Ala His Gly Gly Cys Gly Gly Ser Ala Ala Ala

65 70 75

gggtgtggga ggcctaagct t 261

<210>30

<211>78

<212>PRT

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>30

Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu

1 5 10 15

Cys Leu Pro Trp Leu Gln Glu Gly Ser Ala Cys Gly Gly Leu Thr Asp

20 25 30

Thr Leu Gln Ala Glu Thr Asp Gln Val Glu Asp Glu Lys Ser Ala Leu

35 40 45

Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu Lys Glu Lys Leu Glu Phe

50 55 60

Ile Leu Ala Ala His Gly Gly Cys Gly Gly Ser Ala Ala Ala

65 70 75

<210>31

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>31

cctgggtggg ggcggccgct tctggtggtt gcggtggtct gacc 44

<210>32

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>32

ggtgggaatt caggaggtaa aaagatatcg ggtgtggggc ggcc 44

<210>33

<211>47

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>33

ggtgggaatt caggaggtaa aaaacgatgg cttgcggtgg tctgacc 47

<210>34

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>34

gcttgcggtg gtctgacc 18

<210>35

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>35

ccaccaagct tagcaaccac cgtgtgc 27

<210>36

<211>54

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>36

ccaccaagct tgatatcccc acacccagcg gccgcagaac caccgcaacc accg 54

<210>37

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>37

ccaccaagct taggcctccc acacccagcg gc 32

<210>38

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>38

ggtgggaatt caggaggtaa aaaacgatg 29

<210>39

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>39

ggtgggaatt caggcctatg gctacaggct cc 32

<210>40

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>40

ggtgggaatt catggctaca ggctccc 27

<210>41

<211>59

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>41

gggtctagaa tggctacagg ctcccggacg tccctgctcc tggcttttgg cctgctctg 59

<210>42

<211>58

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>42

cgcaggcctc ggcactgccc tcttgaagcc agggcaggca gagcaggcca aaagccag 58

<210>43

<211>402

<212>DNA

<213> Artificial sequence

<220>

<223> modified bee venom phospholipase A2

<220>

<221>CDS

<222>(1)..(402)

<400>43

atc atc tac cca ggt act ctg tgg tgt ggt cac ggc aac aaa tct tct 48

Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly His Gly Asn Lys Ser Ser

1 5 10 15

ggt ccg aac gaa ctc ggc cgc ttt aaa cac acc gac gca tgc tgt cgc 96

Gly Pro Asn Glu Leu Gly Arg Phe Lys His Thr Asp Ala Cys Cys Arg

20 25 30

acc cag gac atg tgt ccg gac gtc atg tct gct ggt gaa tct aaa cac 144

Thr Gln Asp Met Cys Pro Asp Val Met Ser Ala Gly Glu Ser Lys His

35 40 45

ggg tta act aac acc gct tct cac acg cgt ctc agc tgc gac tgc gac 192

Gly Leu Thr Asn Thr Ala Ser His Thr Arg Leu Ser Cys Asp Cys Asp

50 55 60

gac aaa ttc tac gac tgc ctt aag aac tcc gcc gat acc atc tct tct 240

Asp Lys Phe Tyr Asp Cys Leu Lys Asn Ser Ala Asp Thr Ile Ser Ser

65 70 75 80

tac ttc gtt ggt aaa atg tat ttc aac ctg atc gat acc aaa tgt tac 288

Tyr Phe Val Gly Lys Met Tyr Phe Asn Leu Ile Asp Thr Lys Cys Tyr

85 90 95

aaa ctg gaa cac ccg gta acc ggc tgc ggc gaa cgt acc gaa ggt cgc 336

Lys Leu Glu His Pro Val Thr Gly Cys Gly Glu Arg Thr Glu Gly Arg

100 105 110

tgc ctg cac tac acc gtt gac aaa tct aaa ccg aaa gtt tac cag tgg 384

Cys Leu His Tyr Thr Val Asp Lys Ser Lys Pro Lys Val Tyr Gln Trp

115 120 125

ttc gac ctg cgc aaa tac 402

Phe Asp Leu Arg Lys Tyr

130

<210>44

<211>134

<212>PRT

<213> Artificial sequence

<220>

<223> modified bee venom phospholipase A2

<400>44

Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly His Gly Asn Lys Ser Ser

1 5 10 15

Gly Pro Asn Glu Leu Gly Arg Phe Lys His Thr Asp Ala Cys Cys Arg

20 25 30

Thr Gln Asp Met Cys Pro Asp Val Met Ser Ala Gly Glu Ser Lys His

35 40 45

Gly Leu Thr Asn Thr Ala Ser His Thr Arg Leu Ser Cys Asp Cys Asp

50 55 60

Asp Lys Phe Tyr Asp Cys Leu Lys Asn Ser Ala Asp Thr Ile Ser Ser

65 70 75 80

Tyr Phe Val Gly Lys Met Tyr Phe Asn Leu Ile Asp Thr Lys Cys Tyr

85 90 95

Lys Leu Glu His Pro Val Thr Gly Cys Gly Glu Arg Thr Glu Gly Arg

100 105 110

Cys Leu His Tyr Thr Val Asp Lys Ser Lys Pro Lys Val Tyr Gln Trp

115 120 125

Phe Asp Leu Arg Lys Tyr

130

<210>45

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>45

ccatcatcta cccaggtac 19

<210>46

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>46

cccacaccca gcggccgcgt atttgcgcag gtcg 34

<210>47

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>47

cggtggttct gcggccgcta tcatctaccc aggtac 36

<210>48

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>48

ttagtatttg cgcaggtcg 19

<210>49

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>49

ccggctccat cggtgcag 18

<210>50

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>50

accaccagaa gcggccgcag gggaaacaca tctgcc 36

<210>51

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>51

cggtggttct gcggccgctg gctccatcgg tgcag 35

<210>52

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>52

ttaaggggaa acacatctgc c 21

<210>53

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>53

actagtctag aatgagagtg aaggagaaat atc 33

<210>54

<211>42

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>54

tagcatgcta gcaccgaatt tatctaattc caataattct tg 42

<210>55

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>55

gtagcaccca ccaaggcaaa gctgaaagct acccagctcg agaaactggc a 51

<210>56

<211>48

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>56

caaagctcct attcccactg ccagtttctc gagctgggta gctttcag 48

<210>57

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>57

ttcggtgcta gcggtggctg cggtggtctg accgac 36

<210>58

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>58

gatgctgggc ccttaaccgc aaccaccgtg tgccgcc 37

<210>59

<211>46

<212>PRT

<213> Artificial sequence

<220>

<223> JUN amino acid sequence

<400>59

Cys Gly Gly Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu Lys

1 5 10 15

Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln

20 25 30

Val Ala Gln Leu Lys Gln Lys Val Met Asn His Val Gly Cys

35 40 45

<210>60

<211>46

<212>PRT

<213> Artificial sequence

<220>

<223> FOS amino acid sequence

<400>60

Cys Gly Gly Leu Thr Asp Thr Leu Gln Ala Glu Thr Asp Gln Val Glu

1 5 10 15

Asp Glu Lys Ser Ala Leu Gln Thr Glu Ile Ala Asn Leu Leu Lys Glu

20 25 30

Lys Glu Lys Leu Glu Phe Ile Leu Ala Ala His Gly Gly Cys

35 40 45

<210>61

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>61

ccggaattca tgtgcggtgg tcggatcgcc cgg 33

<210>62

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>62

gtcgctaccc gcggctccgc aaccaacgtg gttcatgac 39

<210>63

<211>50

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>63

gttggttgcg gagccgcgggtagcgacatt gacccttata aagaatttgg 50

<210>64

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>64

cgcgtcccaa gcttctacgg aagcgttgat aggatagg 38

<210>65

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>65

ctagccgcgg gttgcggtgg tcggatcgcc cgg 33

<210>66

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>66

cgcgtcccaa gcttttagca accaacgtgg ttcatgac 38

<210>67

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>67

ccggaattca tggacattga cccttataaa g 31

<210>68

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>68

ccgaccaccg caacccgcgg ctagcggaag cgttgatagg atagg 45

<210>69

<211>47

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>69

ctaatggatc cggtgggggc tgcggtggtc ggatcgcccg gctcgag 47

<210>70

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide primer

<400>70

gtcgctaccc gcggctccgc aaccaacgtg gttcatgac 39

<210>71

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>71

ccggaattca tggacattga cccttataaa g 31

<210>72

<211>48

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>72

ccgaccaccg cagcccccac cggatccatt agtacccacc caggtagc 48

<210>73

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223>oilgonucleotide primer

<400>73

gttggttgcg gagccgcggg tagcgaccta gtagtcagtt atgtc 45

<210>74

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>74

cgcgtcccaa gcttctacgg aagcgttgat aggatagg 38

<210>75

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>75

ctagccgcgg gttgcggtgg tcggatcgcc cgg 33

<210>76

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>76

cgcgtcccaa gcttttagca accaacgtgg ttcatgac 38

<210>77

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>77

ccggaattca tggccacact tttaaggagc 30

<210>78

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>78

cgcgtcccaa gcttttagca accaacgtgg ttcatgac 38

<210>79

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>79

ccggaattca tggacattga cccttataaa g 31

<210>80

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>80

cctagagcca cctttgccac catcttctaa attagtaccc acccaggtag c 51

<210>81

<211>48

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>81

gaagatggtg gcaaaggtgg ctctagggac ctagtagtca gttatgtc 48

<210>82

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>82

cgcgtcccaa gcttctaaac aacagtagtc tccggaag 38

<210>83

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>83

gccgaattcc tagcagctag caccgaattt atctaa 36

<210>84

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>84

ggttaagtcg acatgagagt gaaggagaaa tat 33

<210>85

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>85

taaccgaatt caggaggtaa aaagatatgg 30

<210>86

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>86

gaagtaaagc ttttaaccac cgcaaccacc agaag 35

<210>87

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>87

tcgaatgggc cctcatcttc gtgtgctagt cag 33

<210>88

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> Fos fusion constructs

<400>88

Glu Phe Arg Arg

1

<210>89

<211>183

<212>PRT

<213> hepatitis B Virus

<400>89

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Gly Ser Gln Cys

180

<210>90

<211>183

<212>PRT

<213> hepatitis B Virus

<400>90

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Thr

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Thr Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Cys Val Ile Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Gly Ser Gln Cys

180

<210>91

<211>212

<212>PRT

<213> hepatitis B Virus

<400>91

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>92

<211>212

<212>PRT

<213> hepatitis B Virus

<400>92

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Glu Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Asn Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>93

<211>183

<212>PRT

<213> hepatitis B Virus

<400>93

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Thr Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Cys Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>94

<211>212

<212>PRT

<213> hepatitis B Virus

<400>94

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Val Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>95

<211>212

<212>PRT

<213> hepatitis B Virus

<400>95

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Asp Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Val Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>96

<211>212

<212>PRT

<213> hepatitis B Virus

<400>96

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro Gln

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>97

<211>212

<212>PRT

<213> hepatitis B Virus

<400>97

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Glu Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Lys Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Gly Ser Gln Cys

210

<210>98

<211>183

<212>PRT

<213> hepatitis B Virus

<400>98

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Phe Arg Asp Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Asp Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Ser Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Cys Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>99

<211>183

<212>PRT

<213> hepatitis B Virus

<400>99

Met Asp Ile Asp Pro Tyr Leu Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>100

<211>212

<212>PRT

<213> hepatitis B Virus

<400>100

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Leu Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg His Ala Ile Leu Cys Trp Gly Asp Leu Arg Thr

85 90 95

Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>101

<211>212

<212>PRT

<213> hepatitis B Virus

<400>101

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Asp Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Phe Arg Asp Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Ala Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Gln Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Cys

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>102

<211>183

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic human hepatitis B construct

<400>102

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>103

<211>212

<212>PRT

<213> hepatitis B Virus

<400>103

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Ser

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ile Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pre Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>104

<211>183

<212>PRT

<213> hepatitis B Virus

<400>104

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>105

<211>183

<212>PRT

<213> hepatitis B Virus

<400>105

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>106

<211>183

<212>PRT

<213> hepatitis B Virus

<400>106

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Ala Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Thr Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>107

<211>212

<212>PRT

<213> hepatitis B Virus

<400>107

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Gln Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Tyr Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>108

<211>212

<212>PRT

<213> hepatitis B Virus

<400>108

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>109

<211>212

<212>PRT

<213> hepatitis B Virus

<400>109

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ala Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>110

<211>212

<212>PRT

<213> hepatitis B Virus

<400>110

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Phe Glu Cys Ser Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>111

<211>212

<212>PRT

<213> hepatitis B Virus

<220>

<221>UNSURE

<222>(28)

<223>May be any amino acid

<400>111

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Xaa Asp Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Ile Thr

85 90 95

Leu Ser Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Thr Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Thr Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>112

<211>212

<212>PRT

<213> hepatitis B Virus

<400>112

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Asn Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>113

<211>212

<212>PRT

<213> hepatitis B Virus

<400>113

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Cys Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>114

<211>212

<212>PRT

<213> hepatitis B Virus

<400>114

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Pro Gln Cys

210

<210>115

<211>212

<212>PRT

<213> hepatitis B Virus

<400>115

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Ser Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>116

<211>212

<212>PRT

<213> hepatitis B Virus

<400>116

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Leu Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>117

<211>212

<212>PRT

<213> hepatitis B Virus

<400>117

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Lys Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser GlnCys

210

<210>118

<211>212

<212>PRT

<213> hepatitis B Virus

<400>118

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ala

50 55 60

Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Thr Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>119

<211>183

<212>PRT

<213> hepatitis B Virus

<400>119

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Met Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Tyr Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Thr Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Gln Asp Pro Thr

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Val Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Val Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Gln Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Cys Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>120

<211>183

<212>PRT

<213> hepatitis B Virus

<400>120

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg His Val Phe Leu Cys Trp Gly Asp

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Thr

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>121

<211>212

<212>PRT

<213> hepatitis B Virus

<400>121

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Thr Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>122

<211>212

<212>PRT

<213> hepatitis B Virus

<400>122

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Ile Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>123

<211>183

<212>PRT

<213> hepatitis B Virus

<400>123

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Val

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Ala Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>124

<211>212

<212>PRT

<213> hepatitis B Virus

<400>124

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Asn

85 90 95

Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Val Ser Arg Asp

100 105 110

Leu Val Val Gly Tyr Val Asn Thr Thr Val Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>125

<211>183

<212>PRT

<213> hepatitis B Virus

<400>125

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Thr Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>126

<211>212

<212>PRT

<213> hepatitis B Virus

<400>126

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Ala Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Ile Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>127

<211>212

<212>PRT

<213> hepatitis B Virus

<400>127

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Thr Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>128

<211>212

<212>PRT

<213> hepatitis B Virus

<400>128

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Arg Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Thr Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>129

<211>212

<212>PRT

<213> hepatitis B Virus

<400>129

Met Gln Leu Phe His Leu Cys Leu Val Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ala

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>130

<211>212

<212>PRT

<213> hepatitis B Virus

<400>130

Met Gln Leu Phe His Leu Cys Leu Ile Ile Ser Cys Ser Cys Pro Thr

1 5 10 15

Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Gly Met Asp Ile

20 25 30

Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu

35 40 45

Pro Ser Ala Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser

50 55 60

Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His

65 70 75 80

His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp Leu Met Thr

85 90 95

Leu Ala Thr Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp

100 105 110

Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln

115 120 125

Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val

130 135 140

Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr

165 170 175

Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

180 185 190

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

195 200 205

Glu Ser Gln Cys

210

<210>131

<211>183

<212>PRT

<213> hepatitis B Virus

<400>131

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ala Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Glu Ser Gln Cys

180

<210>132

<211>183

<212>PRT

<213> hepatitis B Virus

<400>132

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ile

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Cys Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

145 150 155 160

Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

165 170 175

Gln Ser Arg Gly Ser Gln Cys

180

<210>133

<211>3221

<212>DNA

<213> hepatitis B Virus

<220>

<221>CDS

<222>(1901)..(2458)

<400>133

ttccactgcc ttccaccaag ctctgcagga ccccagagtc aggggtctgt attttcctgc 60

tggtggctcc agttcaggaa cagtaaaccc tgctccgaat attgcctctc acatctcgtc 120

aatctccgcg aggactgggg accctgtgac gaacatggag aacatcacat caggattcct 180

aggacccctg ctcgtgttac aggcggggtt tttattgttg acaagaatcc tcacaatacc 240

gcagagtcta gactcgtggt ggacttctct caattttata gggggatcac ccgtgtgtct 300

tggccaaaat tcgcagtccc caacctccaa tcactcacca acctcctgtc ctccaatttg 360

tcctggttat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420

atgcctcatc ttcttattgg ttcttctgga ttatcaaggt atgttgcccg tttgtcctct 480

aattccagga tcaacaacaa ccagtacggg accatgcaaa acctgcacga ctcctgctca 540

aggcaactct atgtttccct catgttgctg tacaaaacct acggttggaa attgcacctg 600

tattcccatc ccatcgtcct gggctttcgc aaaataccta tgggagtggg cctcagtccg 660

tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720

tgtttggctt tcagctatat ggatgatgtg gtattggggg ccaagtctgt acagcatcgt 780

gagtcccttt ataccgctgt taccaatttt cttttgtctc tgggtataca tttaaaccct 840

aacaaaacaa aaagatgggg ttattcccta aacttcatgg gttacataat tggaagttgg 900

ggaacattgc cacaggatca tattgtacaa aagatcaaac actgttttag aaaacttcct 960

gttaacaggc ctattgattg gaaagtatgt caaagaattg tgggtctttt gggctttgct 1020

gctccattta cacaatgtgg atatcctgcc ttaatgcctt tgtatgcatg tatacaggct 1080

aaacaggctt tcactttctc gccaacttac aaggcctttc taagtaaaca gtacatgaac 1140

ctttaccccg ttgctcggca acggcctggt ctgtgccaag tgtttgctga cgcaaccccc 1200

actggttggg gcttggccat aggccatcag cgcatgagtg gaacctttgt ggctcctctg 1260

ccgatccata ctgcggaact cctagccgct tgtattgctc gcagccggtc tggagcaaag 1320

ctcatcggaa ctgacaattc tgtcgtcctc tcgcggaaat atacatcgtt tccatggctg 1380

ctaggctgta ctgccaactg gatccttcgc gggacgtcct ttgtttacgt cccgtcggcg 1440

ctgaatcccg cggacgaccc ctctcggggc cgcttgggac tctatcgtcc ccttctccgt 1500

ctgccgttcc agccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgtgcct 1560

tctcatctgc cggtccgtgt gcacttcgct tcacctctgc acgttgcatg gagaccaccg 1620

tgaacgccca tcagatcctg cccaaggtct tacataagag gactcttgga ctcccagcaa 1680

tgtcaacgac cgaccttgag gcctacttca aagactgtgt gtttaaggac tgggaggagc 1740

tgggggagga gattaggtta aaggtctttg tattaggagg ctgtaggcat aaattggtct 1800

gcgcaccagc accatgcaac tttttcacct ctgcctaatc atctcttgta catgtcccac 1860

tgttcaagcc tccaagctgt gccttgggtg gctttggggc atg gac att gac cct 1915

Met Asp Ile Asp Pro

1 5

tat aaa gaa ttt gga gct act gtg gag tta ctc tcg ttt ttg cct tct 1963

Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser

10 15 20

gac ttc ttt cct tcc gtc aga gat ctc cta gac acc gcc tca gct ctg 2011

Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu

25 30 35

tat cga gaa gcc tta gag tct cct gag cat tgc tca cct cac cat act 2059

Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr

40 45 50

gca ctc agg caa gcc att ctc tgc tgg ggg gaa ttg atg act cta gct 2107

Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala

55 60 65

acc tgg gtg ggt aat aat ttg gaa gat cca gca tcc agg gat cta gta 2155

Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala Ser Arg Asp Leu Val

70 75 80 85

gtc aat tat gtt aat act aac atg ggt tta aag atc agg caa cta ttg 2203

Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu

90 95 100

tgg ttt cat ata tct tgc ctt act ttt gga aga gag act gta ctt gaa 2251

Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu

105 110 115

tat ttg gtc tct ttc gga gtg tgg att cgc act cct cca gcc tat aga 2299

Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg

120 125 130

cca cca aat gcc cct atc tta tca aca ctt ccg gaa act act gtt gtt 2347

Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val

135 140 145

aga cga cgg gac cga ggc agg tcc cct aga aga aga act ccc tcg cct 2395

Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro

150 155 160 165

cgc aga cgc aga tct caa tcg ccg cgt cgc aga aga tct caa tct cgg 2443

Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg

170 175 180

gaa tct caa tgt tag tat tccttgg actcataagg tgggaaactt tactgggctt 2498

Glu Ser Gln Cys

185

tattcctcta cagtacctat ctttaatcct gaatggcaaa ctccttcctt tcctaagatt 2558

catttacaag aggacattat tgataggtgt caacaatttg tgggccctct cactgtaaat 2618

gaaaagagaa gattgaaatt aattatgcct gctagattct atcctaccca cactaaatat 2678

ttgcccttag acaaaggaat taaaccttat tatccagatc aggtagttaa tcattacttc 2738

caaaccagac attatttaca tactctttgg aaggctggta ttctatataa gagggaaacc 2798

acacgtagcg catcattttg cgggtcacca tattcttggg aacaagagct acagcatggg 2858

aggttggtca ttaaaacctc gcaaaggcat ggggacgaat ctttctgttc ccaaccctct 2918

gggattcttt cccgatcatc agttggaccc tgcattcgga gccaactcaa acaatccaga 2978

ttgggacttc aaccccatca aggaccactg gccagcagcc aaccaggtag gagtgggagc 3038

attcgggcca gggctcaccc ctccacacgg cggtattttg gggtggagcc ctcaggctca 3098

gggcatattg accacagtgt caacaattcc tcctcctgcc tccaccaatc ggcagtcagg 3158

aaggcagcct actcccatct ctccacctct aagagacagt catcctcagg ccatgcagtg 3218

gaa 3221

<210>134

<211>185

<212>PRT

<213> hepatitis B Virus

<400>134

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys

85 90 95

Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg

145 150 155 160

Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg

165 170 175

Arg Ser Gln Ser Arg Glu Ser Gln Cys

180 185

<210>135

<211>188

<212>PRT

<213> woodchuck hepatitis B Virus

<400>135

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu

1 5 10 15

Asn Phe Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp

20 25 30

Thr Ala Thr Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys

35 40 45

Ser Pro His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Asp Glu

50 55 60

Leu Thr Lys Leu Ile Ala Trp Met Ser Ser Asn Ile Thr Ser Glu Gln

65 70 75 80

Val Arg Thr Ile Ile Val Asn His Val Asn Asp Thr Trp Gly Leu Lys

85 90 95

Val Arg Gln Ser Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln

100 105 110

His Thr Val Gln Glu Phe Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Ala Pro Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu His Thr Val Ile Arg Arg Arg Gly Gly Ala Arg Ala Ser Arg Ser

145 150 155 160

Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro

165 170 175

Arg Arg Arg Arg Ser Gln Ser Pro Ser Thr Asn Cys

180 185

<210>136

<211>217

<212>PRT

<213> Disong tree hepatitis B Virus

<400>136

Met Tyr Leu Phe His Leu Cys Leu Val Phe Ala Cys Val Pro Cys Pro

1 5 10 15

Thr Val Gln Ala Ser Lys Leu Cys Leu Gly Trp Leu Trp Asp Met Asp

20 25 30

Ile Asp Pro Tyr Lys Glu Phe Gly Ser Ser Tyr Gln Leu Leu Asn Phe

35 40 45

Leu Pro Leu Asp Phe Phe Pro Asp Leu Asn Ala Leu Val Asp Thr Ala

50 55 60

Ala Ala Leu Tyr Glu Glu Glu Leu Thr Gly Arg Glu His Cys Ser Pro

65 70 75 80

His His Thr Ala Ile Arg Gln Ala Leu Val Cys Trp Glu Glu Leu Thr

85 90 95

Arg Leu Ile Thr Trp Met Ser Glu Asn Thr Thr Glu Glu Val Arg Arg

100 105 110

Ile Ile Val Asp His Val Asn Asn Thr Trp Gly Leu Lys Val Arg Gln

115 120 125

Thr Leu Trp Phe His Leu Ser Cys Leu Thr Phe Gly Gln His Thr Val

130 135 140

Gln Glu Phe Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Ala Pro

145 150 155 160

Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu His Thr

165 170 175

Val Ile Arg Arg Arg Gly Gly Ser Arg Ala Ala Arg Ser Pro Arg Arg

180 185 190

Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg

195 200 205

Arg Ser Gln Ser Pro Ala Ser Asn Cys

210 215

<210>137

<211>262

<212>PRT

<213> hepatitis B virus of wild goose

<400>137

Met Asp Val Asn Ala Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro

1 5 10 15

Asp Asp Phe Phe Pro Lys Ile Glu Asp Leu Val Arg Asp Ala Lys Asp

20 25 30

Ala Leu Glu Pro Tyr Trp Lys Ser Asp Ser Ile Lys Lys His Val Leu

35 40 45

Ile Ala Thr His Phe Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr

50 55 60

Gln Gly Met His Glu Ile Ala Glu Ala Ile Arg Ala Val Ile Pro Pro

65 70 75 80

Thr Thr Ala Pro Val Pro Ser Gly Tyr Leu Ile Gln His Asp Glu Ala

85 90 95

Glu Glu Ile Pro Leu Gly Asp Leu Phe Lys Glu Gln Glu Glu Arg Ile

100 105 110

Val Ser Phe Gln Pro Asp Tyr Pro Ile Thr Ala Arg Ile His Ala His

115 120 125

Leu Lys Ala Tyr Ala Lys Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg

130 135 140

Arg Leu Leu Trp Trp His Tyr Asn Cys Leu Leu Trp Gly Glu Ala Thr

145 150 155 160

Val Thr Asn Tyr Ile Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu

165 170 175

Lys Tyr Arg Gly Arg Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro

180 185 190

Ile Gln Val Ala Gln Gly Gly Arg Lys Thr Ser Thr Ala Thr Arg Lys

195 200 205

Pro Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr Val Val

210 215 220

Tyr Gly Arg Arg Arg Ser Lys Ser Arg Glu Arg Arg Ala Ser Ser Pro

225 230 235 240

Gln Arg Ala Gly Ser Pro Leu Pro Arg Ser Ser Ser Ser His His Arg

245 250 255

Ser Pro Ser Pro Arg Lys

260

<210>138

<211>305

<212>PRT

<213>Duck hepatitis virus

<400>138

Met Trp Asp Leu Arg Leu His Pro Ser Pro Phe Gly Ala Ala Cys Gln

1 5 10 15

Gly Ile Phe Thr Ser Ser Leu Leu Leu Phe Leu Val Thr Val Pro Leu

20 25 30

Val Cys Thr Ile Val Tyr Asp Ser Cys Leu Cys Met Asp Ile Asn Ala

35 40 45

Ser Arg Ala Leu Ala Asn Val Tyr Asp Leu Pro Asp Asp Phe Phe Pro

50 55 60

Lys Ile Asp Asp Leu Val Arg Asp Ala Lys Asp Ala Leu Glu Pro Tyr

65 70 75 80

Trp Arg Asn Asp Ser Ile Lys Lys His Val Leu Ile Ala Thr His Phe

85 90 95

Val Asp Leu Ile Glu Asp Phe Trp Gln Thr Thr Gln Gly Met His Glu

100 105 110

Ile Ala Glu Ala Leu Arg Ala Ile Ile Pro Ala Thr Thr Ala Pro Val

115 120 125

Pro Gln Gly Phe Leu Val Gln His Glu Glu Ala Glu Glu Ile Pro Leu

130 135 140

Gly Glu Leu Phe Arg Tyr Gln Glu Glu Arg Leu Thr Asn Phe Gln Pro

145 150 155 160

Asp Tyr Pro Val Thr Ala Arg Ile His Ala His Leu Lys Ala Tyr Ala

165 170 175

Lys Ile Asn Glu Glu Ser Leu Asp Arg Ala Arg Arg Leu Leu Trp Trp

180 185 190

His Tyr Asn Cys Leu Leu Trp Gly Glu Pro Asn Val Thr Asn Tyr Ile

195 200 205

Ser Arg Leu Arg Thr Trp Leu Ser Thr Pro Glu Lys Tyr Arg Gly Lys

210 215 220

Asp Ala Pro Thr Ile Glu Ala Ile Thr Arg Pro Ile Gln Val Ala Gln

225 230 235 240

Gly Gly Arg Asn Lys Thr Gln Gly Val Arg Lys Ser Arg Gly Leu Glu

245 250 255

Pro Arg Arg Arg Arg Val Lys Thr Thr Ile Val Tyr Gly Arg Arg Arg

260 265 270

Ser Lys Ser Arg Glu Arg Arg Ala Pro Thr Pro Gln Arg Ala Gly Ser

275 280 285

Pro Leu Pro Arg Thr Ser Arg Asp His His Arg Ser Pro Ser Pro Arg

290 295 300

Glu

305

<210>139

<211>212

<212>PRT

<213> Haemophilus influenzae

<400>139

Met Lys Lys Thr Leu Leu Gly Ser Leu Ile Leu Leu Ala Phe Ala Gly

1 5 10 15

Asn Val Gln Ala Ala Ala Asn Ala Asp Thr Ser Gly Thr Val Thr Phe

20 25 30

Phe Gly Lys Val Val Glu Asn Thr Cys Gln Val Asn Gln Asp Ser Glu

35 40 45

Tyr Glu Cys Asn Leu Asn Asp Val Gly Lys Asn His Leu Ser Gln Gln

50 55 60

Gly Tyr Thr Ala Met Gln Thr Pro Phe Thr Ile Thr Leu Glu Asn Cys

65 70 75 80

Asn Val Thr Thr Thr Asn Asn Lys Pro Lys Ala Thr Lys Val Gly Val

85 90 95

Tyr Phe Tyr Ser Trp Glu Ile Ala Asp Lys Asp Asn Lys Tyr Thr Leu

100 105 110

Lys Asn Ile Lys Glu Asn Thr Gly Thr Asn Asp Ser Ala Asn Lys Val

115 120 125

Asn Ile Gln Leu Leu Glu Asp Asn Gly Thr Ala Glu Ile Lys Val Val

130 135 140

Gly Lys Thr Thr Thr Asp Phe Thr Ser Glu Asn His Asn Gly Ala Gly

145 150 155 160

Ala Asp Pro Val Ala Thr Asn Lys His Ile Ser Ser Leu Thr Pro Leu

165 170 175

Asn Asn Gln Asn Ser Ile Asn Leu His Tyr Ile Ala Gln Tyr Tyr Ala

180 185 190

Thr Gly Val Ala Glu Ala Gly Lys Val Pro Ser Ser Val Asn Ser Gln

195 200 205

Ile Ala Tyr Glu

210

<210>140

<211>139

<212>PRT

<213> Pseudomonas stutzeri

<400>140

Met Lys Ala Gln Met Gln Lys Gly Phe Thr Leu Ile Glu Leu Met Ile

1 5 10 15

Val Val Ala Ile Ile Gly Ile Leu Ala Ala Ile Ala Leu Pro Ala Tyr

20 25 30

Gln Asp Tyr Thr Val Arg Ser Asn Ala Ala Ala Ala Leu Ala Glu Ile

35 40 45

Thr Pro Gly Lys Ile Gly Phe Glu Gln Ala Ile Asn Glu Gly Lys Thr

50 55 60

Pro Ser Leu Thr Ser Thr Asp Glu Gly Tyr Ile Gly Ile Thr Asp Ser

65 70 75 80

Thr Ser Tyr Cys Asp Val Asp Leu Asp Thr Ala Ala Asp Gly His Ile

85 90 95

Glu Cys Thr Ala Lys Gly Gly Asn Ala Gly Lys Phe Asp Gly Lys Thr

100 105 110

Ile Thr Leu Asn Arg Thr Ala Asp Gly Glu Trp Ser Cys Ala Ser Thr

115 120 125

Leu Asp Ala Lys Tyr Lys Pro Gly Lys Cys Ser

130 135

<210>141

<211>59

<212>PRT

<213> Bacillus crescentus

<400>141

Met Thr Lys Phe Val Thr Arg Phe Leu Lys Asp Glu Ser Gly Ala Thr

1 5 10 15

Ala Ile Glu Tyr Gly Leu Ile Val Ala Leu Ile Ala Val Val Ile Val

20 25 30

Thr Ala Val Thr Thr Leu Gly Thr Asn Leu Arg Thr Ala Phe Thr Lys

35 40 45

Ala Gly Ala Ala Val Ser Thr Ala Ala Gly Thr

50 55

<210>142

<211>173

<212>PRT

<213> Escherichia coli

<400>142

Met Ala Val Val Ser Phe Gly Val Asn Ala Ala Pro Thr Ile Pro Gln

1 5 10 15

Gly Gln Gly Lys Val Thr Phe Asn Gly Thr Val Val Asp Ala Pro Cys

20 25 30

Ser Ile Ser Gln Lys Ser Ala Asp Gln Ser Ile Asp Phe Gly Gln Leu

35 40 45

Ser Lys Ser Phe Leu Glu Ala Gly Gly Val Ser Lys Pro Met Asp Leu

50 55 60

Asp Ile Glu Leu Val Asn Cys Asp Ile Thr Ala Phe Lys Gly Gly Asn

65 70 75 80

Gly Ala Gln Lys Gly Thr Val Lys Leu Ala Phe Thr Gly Pro Ile Val

85 90 95

Asn Gly His Ser Asp Glu Leu Asp Thr Asn Gly Gly Thr Gly Thr Ala

100 105 110

Ile Val Val Gln Gly Ala Gly Lys Asn Val Val Phe Asp Gly Ser Glu

115 120 125

Gly Asp Ala Asn Thr Leu Lys Asp Gly Glu Asn Val Leu His Tyr Thr

130 135 140

Ala Val Val Lys Lys Ser Ser Ala Val Gly Ala Ala Val Thr Glu Gly

145 150 155 160

Ala Phe Ser Ala Val Ala Asn Phe Asn Leu Thr Tyr Gln

165 170

<210>143

<211>173

<212>PRT

<213> Escherichia coli

<400>143

Met Ala Val Val Ser Phe Gly Val Asn Ala Ala Pro Thr Ile Pro Gln

1 5 10 15

Gly Gln Gly Lys Val Thr Phe Asn Gly Thr Val Val Asp Ala Pro Cys

20 25 30

Ser Ile Ser Gln Lys Ser Ala Asp Gln Ser Ile Asp Phe Gly Gln Leu

35 40 45

Ser Lys Ser Phe Leu Glu Ala Gly Gly Val Ser Lys Pro Met Asp Leu

50 55 60

Asp Ile Glu Leu Val Asn Cys Asp Ile Thr Ala Phe Lys Gly Gly Asn

65 70 75 80

Gly Ala Gln Lys Gly Thr Val Lys Leu Ala Phe Thr Gly Pro Ile Val

85 90 95

Asn Gly His Ser Asp Glu Leu Asp Thr Asn Gly Gly Thr Gly Thr Ala

100 105 110

Ile Val Val Gln Gly Ala Gly Lys Asn Val Val Phe Asp Gly Ser Glu

115 120 125

Gly Asp Ala Asn Thr Leu Lys Asp Gly Glu Asn Val Leu His Tyr Thr

130 135 140

Ala Val Val Lys Lys Ser Ser Ala Val Gly Ala Ala Val Thr Glu Gly

145 150 155 160

Ala Phe Ser Ala Val Ala Asn Phe Asn Lau Thr Tyr Gln

165 170

<210>144

<211>172

<212>PRT

<213> Escherichia coli

<400>144

Met Ala Val Val Ser Phe Gly Val Asn Ala Ala Pro Thr Thr Pro Gln

1 5 10 15

Gly Gln Gly Arg Val Thr Phe Asn Gly Thr Val Val Asp Ala Pro Cys

20 25 30

Ser Ile Ser Gln Lys Ser Ala Asp Gln Ser Ile Asp Phe Gly Gln Leu

35 40 45

Ser Lys Ser Phe Leu Ala Asn Asp Gly Gln Ser Lys Pro Met Asn Leu

50 55 60

Asp Ile Glu Leu Val Asn Cys Asp Ile Thr Ala Phe Lys Asn Gly Asn

65 70 75 80

Ala Lys Thr Gly Ser Val Lys Leu Ala Phe Thr Gly Pro Thr Val Ser

85 90 95

Gly His Pro Ser Glu Leu Ala Thr Asn Gly Gly Pro Gly Thr Ala Ile

100 105 110

Met Ile Gln Ala Ala Gly Lys Asn Val Pro Phe Asp Gly Thr Glu Gly

115 120 125

Asp Pro Asn Leu Leu Lys Asp Gly Asp Asn Val Leu His Tyr Thr Thr

130 135 140

Val Gly Lys Lys Ser Ser Asp Gly Asn Ala Gln Ile Thr Glu Gly Ala

145 150 155 160

Phe Ser Gly Val Ala Thr Phe Asn Leu Ser Tyr Gln

165 170

<210>145

<211>853

<212>DNA

<213> Escherichia coli

<220>

<221>CDS

<222>(281)..(829)

<400>145

acgtttctgt ggctcgacgc atcttcctca ttcttctctc caaaaaccac ctcatgcaat 60

ataaacatct ataaataaag ataacaaata gaatattaag ccaacaaata aactgaaaaa 120

gtttgtccgc gatgctttac ctctatgagt caaaatggcc ccaatgtttc atcttttggg 180

ggaaactgtg cagtgttggc agtcaaactc gttgacaaac aaagtgtaca gaacgactgc 240

ccatgtcgat ttagaaatag ttttttgaaa ggaaagcagc atg aaa att aaa act 295

Met Lys Ile Lys Thr

1 5

ctg gca atc gtt gtt ctg tcg gct ctg tcc ctc agt tct acg acg gct 343

Leu Ala Ile Val Val Leu Ser Ala Leu Ser Leu Ser Ser Thr Thr Ala

10 15 20

ctg gcc gct gcc acg acg gtt aat ggt ggg acc gtt cac ttt aaa ggg 391

Leu Ala Ala Ala Thr Thr Val Asn Gly Gly Thr Val His Phe Lys Gly

25 30 35

gaa gtt gtt aac gcc gct tgc gca gtt gat gca ggc tct gtt gat caa 439

Glu Val Val Asn Ala Ala Cys Ala Val Asp Ala Gly Ser Val Asp Gln

40 45 50

acc gtt cag tta gga cag gtt cgt acc gca tcg ctg gca cag gaa gga 487

Thr Val Gln Leu Gly Gln Val Arg Thr Ala Ser Leu Ala Gln Glu Gly

55 60 65

gca acc agt tct gct gtc ggt ttt aac att cag ctg aat gat tgc gat 535

Ala Thr Ser Ser Ala Val Gly Phe Asn Ile Gln Leu Asn Asp Cys Asp

70 75 80 85

acc aat gtt gca tct aaa gcc gct gtt gcc ttt tta ggt acg gcg att 583

Thr Asn Val Ala Ser Lys Ala Ala Val Ala Phe Leu Gly Thr Ala Ile

90 95 100

gat gcg ggt cat acc aac gtt ctg gct ctg cag agt tca gct gcg ggt 631

Asp Ala Gly His Thr Asn Val Leu Ala Leu Gln Ser Ser Ala Ala Gly

105 110 115

agc gca aca aac gtt ggt gtg cag atc ctg gac aga acg ggt gct gcg 679

Ser Ala Thr Asn Val Gly Val Gln Ile Leu Asp Arg Thr Gly Ala Ala

120 125 130

ctg acg ctg gat ggt gcg aca ttt agt tca gaa aca acc ctg aat aac 727

Leu Thr Leu Asp Gly Ala Thr Phe Ser Ser Glu Thr Thr Leu Asn Asn

135 140 145

gga acc aat acc att ccg ttc cag gcg cgt tat ttt gca acc ggg gcc 775

Gly Thr Asn Thr Ile Pro Phe Gln Ala Arg Tyr Phe Ala Thr Gly Ala

150 155 160 165

gca acc ccg ggt gct gct aat gcg gat gcg acc ttc aag gtt cag tat 823

Ala Thr Pro Gly Ala Ala Asn Ala Asp Ala Thr Phe Lys Val Gln Tyr

170 175 180

caa taa cctacctagg ttcagggacg ttca 853

Gln

<210>146

<211>182

<212>PRT

<213> Escherichia coli

<400>146

Met Lys Ile Lys Thr Leu Ala Ile Val Val Leu Ser Ala Leu Ser Leu

1 5 10 15

Ser Ser Thr Thr Ala Leu Ala Ala Ala Thr Thr Val Asn Gly Gly Thr

20 25 30

Val His Phe Lys Gly Glu Val Val Asn Ala Ala Cys Ala Val Asp Ala

35 40 45

Gly Ser Val Asp Gln Thr Val Gln Leu Gly Gln Val Arg Thr Ala Ser

50 55 60

Leu Ala Gln Glu Gly Ala Thr Ser Ser Ala Val Gly Phe Asn Ile Gln

65 70 75 80

Leu Asn Asp Cys Asp Thr Asn Val Ala Ser Lys Ala Ala Val Ala Phe

85 90 95

Leu Gly Thr Ala Ile Asp Ala Gly His Thr Asn Val Leu Ala Leu Gln

100 105 110

Ser Ser Ala Ala Gly Ser Ala Thr Asn Val Gly Val Gln Ile Leu Asp

115 120 125

Arg Thr Gly Ala Ala Leu Thr Leu Asp Gly Ala Thr Phe Ser Ser Glu

130 135 140

Thr Thr Leu Asn Asn Gly Thr Asn Thr Ile Pro Phe Gln Ala Arg Tyr

145 150 155 160

Phe Ala Thr Gly Ala Ala Thr Pro Gly Ala Ala Asn Ala Asp Ala Thr

165 170 175

Phe Lys Val Gln Tyr Gln

180

<210>147

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223> FLAG peptide

<400>147

Cys Gly Gly Asp Tyr Lys Asp Asp Asp Asp Lys

1 5 10

<210>148

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>148

ccggaattca tggacattga cccttataaa g 31

<210>149

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>149

gtgcagtatg gtgaggtgag gaatgctcag gagactc 37

<210>150

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>150

gsgtctcctg agcattcctc acctcaccat actgcac 37

<210>151

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>151

cttccaaaag tgagggaaga aatgtgaaac cac 33

<210>152

<211>47

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>152

cgcgtcccaa gcttctaaac aacagtagtc tccggaagcg ttgatag 47

<210>153

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>153

gtggtttcac atttcttccc tcacttttgg aag 33

<210>154

<211>281

<212>PRT

<213>Saccharomyces cerevisiae

<400>154

Met Ser Glu Tyr Gln Pro Ser Leu Phe Ala Leu Asn Pro Met Gly Phe

1 5 10 15

Ser Pro Leu Asp Gly Ser Lys Ser Thr Asn Glu Asn Val Ser Ala Ser

20 25 30

Thr Ser Thr Ala Lys Pro Met Val Gly Gln Leu Ile Phe Asp Lys Phe

35 40 45

Ile Lys Thr Glu Glu Asp Pro Ile Ile Lys Gln Asp Thr Pro Ser Asn

50 55 60

Leu Asp Phe Asp Phe Ala Leu Pro Gln Thr Ala Thr Ala Pro Asp Ala

65 70 75 80

Lys Thr Val Leu Pro Ile Pro Glu Leu Asp Asp Ala Val Val Glu Ser

85 90 95

Phe Phe Ser Ser Ser Thr Asp Ser Thr Pro Met Phe Glu Tyr Glu Asn

100 105 110

Leu Glu Asp Asn Ser Lys Glu Trp Thr Ser Leu Phe Asp Asn Asp Ile

115 120 125

Pro Val Thr Thr Asp Asp Val Ser Leu Ala Asp Lys Ala Ile Glu Ser

130 135 140

Thr Glu Glu Val Ser Leu Val Pro Ser Asn Leu Glu Val Ser Thr Thr

145 150 155 160

Ser Phe Leu Pro Thr Pro Val Leu Glu Asp Ala Lys Leu Thr Gln Thr

165 170 175

Arg Lys Val Lys Lys Pro Asn Ser Val Val Lys Lys Ser His His Val

180 185 190

Gly Lys Asp Asp Glu Ser Arg Leu Asp His Leu Gly Val Val Ala Tyr

195 200 205

Asn Arg Lys Gln Arg Ser Ile Pro Leu Ser Pro Ile Val Pro Glu Ser

210 215 220

Ser Asp Pro Ala Ala Leu Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg

225 230 235 240

Arg Ser Arg Ala Arg Lys Leu Gln Arg Met Lys Gln Leu Glu Asp Lys

245 250 255

Val Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala

260 265 270

Arg Leu Lys Lys Leu Val Gly Glu Arg

275 280

<210>155

<211>181

<212>PRT

<213> Escherichia coli

<400>155

Met Lys Ile Lys Thr Leu Ala Ile Val Val Leu Ser Ala Leu Ser Leu

1 5 10 15

Ser Ser Thr Ala Ala Leu Ala Ala Ala Thr Thr Val Asn Gly Gly Thr

20 25 30

Val His Phe Lys Gly Glu Val Val Asn Ala Ala Cys Ala Val Asp Ala

35 40 45

Gly Ser Val Asp Gln Thr Val Gln Leu Gly Gln Val Arg Thr Ala Ser

50 55 60

Leu Ala Gln Glu Gly Ala Thr Ser Ser Ala Val Gly Phe Asn Ile Gln

65 70 75 80

Leu Asn Asp Cys Asp Thr Asn Val Ala Ser Lys Ala Ala Val Ala Phe

85 90 95

Leu Gly Thr Ala Ile Asp Ala Gly His Thr Asn Val Leu Ala Leu Gln

100 105 110

Ser Ser Ala Ala Gly Ser Ala Thr Asn Val Gly Val Gln Ile Leu Asp

115 120 125

Arg Thr Gly Ala Ala Leu Thr Leu Asp Gly Ala Thr Phe Ser Ser Glu

130 135 140

Thr Thr Leu Asn Asn Gly Thr Asn Thr Ile Pro Phe Gln Ala Arg Tyr

145 150 155 160

Phe Ala Gly Ala Ala Thr Pro Gly Ala Ala Asn Ala Asp Ala Thr Phe

165 170 175

Lys Val Gln Tyr Gln

180

<210>156

<211>447

<212>DNA

<213> hepatitis B

<220>

<221>CDS

<222>(1)..(447)

<400>156

atg gac att gac cct tat aaa gaa ttt gga gct act gtg gag tta ctc 48

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

tcg ttt ttg cct tct gac ttc ttt cct tcc gta cga gat ctt ata gat 96

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

acc gcc gca gct ctg tat cgg gat gcc tta gag tct cct gag cat tgt 144

Thr Ala Ala Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys

35 40 45

tca cct cac cat act gca ctc agg caa gca att ctt tgc tgg gga gac 192

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp

50 55 60

tta atg act cta gct acc tgg gtg ggt act aat tta gaa gat cca gca 240

Leu Met Thr Leu Ala Thr Trp Val Gly Thr Asn Leu Glu Asp Pro Ala

65 70 75 80

tct agg gac cta gta gtc agt tat gtc aac act aat gtg ggc cta aag 288

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys

85 90 95

ttc aga caa tta ttg tgg ttt cac att tct tgt ctc act ttt gga aga 336

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

gaa acg gtt cta gag tat ttg gtc tct ttt gga gtg tgg att cgc act 384

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

cct cca gcc tat aga cca cca aat gcc cct atc cta tca acg ctt ccg 432

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

gag act act gtt gtt 447

Glu Thr Thr Val Val

145

<210>157

<211>149

<212>PRT

<213> hepatitis B

<400>157

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ala Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Thr Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Val Gly Leu Lys

85 90 95

Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val

145

<210>158

<211>152

<212>PRT

<213> hepatitis B

<400>158

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ala Ala Leu Tyr Arg Asp Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Asp

50 55 60

Leu Met Thr Leu Ala Thr Trp Val Gly Thr Asn Leu Glu Asp Gly Gly

65 70 75 80

Lys Gly Gly Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Val

85 90 95

Gly Leu Lys Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr

100 105 110

Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp

115 120 125

Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser

130 135 140

Thr Leu Pro Glu Thr Thr Val Val

145 150

<210>159

<211>132

<212>PRT

<213> bacteriophage Q β

<400>159

Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly Lys

1 5 10 15

Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly Val

20 25 30

Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg Val

35 40 45

Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys Val

50 55 60

Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser Cys

65 70 75 80

Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser Phe

85 90 95

Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu Leu

100 105 110

Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln Leu

115 120 125

Asn Pro Ala Tyr

130

<210>160

<211>129

<212>PRT

<213> bacteriophage R17

<400>160

Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly

1 5 10 15

Asn Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp

20 25 30

Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val

35 40 45

Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val

50 55 60

Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala

65 70 75 80

Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe Ala

85 90 95

Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu

100 105 110

Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile

115 120 125

Tyr

<210>161

<211>130

<212>PRT

<213> phage fr

<400>161

Met Ala Ser Asn Phe Glu Glu Phe Val Leu Val Asp Asn Gly Gly Thr

1 5 10 15

Gly Asp Val Lys Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu

20 25 30

Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser

35 40 45

Val Arg Gln Ser Ser Ala Asn Asn Arg Lys Tyr Thr Val Lys Val Glu

50 55 60

Val Pro Lys Val Ala Thr Gln Val Gln Gly Gly Val Glu Leu Pro Val

65 70 75 80

Ala Ala Trp Arg Ser Tyr Met Asn Met Glu Leu Thr Ile Pro Val Phe

85 90 95

Ala Thr Asn Asp Asp Cys Ala Leu Ile Val Lys Ala Leu Gln Gly Thr

100 105 110

Phe Lys Thr Gly Asn Pro Ile Ala Thr Ala Ile Ala Ala Asn Ser Gly

115 120 125

Ile Tyr

130

<210>162

<211>130

<212>PRT

<213> phage GA

<400>162

Met Ala Thr Leu Arg Ser Phe Val Leu Val Asp Asn Gly Gly Thr Gly

1 5 10 15

Asn Val Thr Val Val Pro Val Ser Asn Ala Asn Gly Val Ala Glu Trp

20 25 30

Leu Ser Asn Asn Ser Arg Ser Gln Ala Tyr Arg Val Thr Ala Ser Tyr

35 40 45

Arg Ala Ser Gly Ala Asp Lys Arg Lys Tyr Ala Ile Lys Leu Glu Val

50 55 60

Pro Lys Ile Val Thr Gln Val Val Asn Gly Val Glu Leu Pro Gly Ser

65 70 75 80

Ala Trp Lys Ala Tyr Ala Ser Ile Asp Leu Thr Ile Pro Ile Phe Ala

85 90 95

Ala Thr Asp Asp Val Thr Val Ile Ser Lys Ser Leu Ala Gly Leu Phe

100 105 110

Lys Val Gly Asn Pro Ile Ala Glu Ala Ile Ser Ser Gln Ser Gly Phe

115 120 125

Tyr Ala

130

<210>163

<211>132

<212>PRT

<213> phage SP

<400>163

Met Ala Lys Leu Asn Gln Val Thr Leu Ser Lys Ile Gly Lys Asn Gly

1 5 10 15

Asp Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn Gly

20 25 30

Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg

35 40 45

Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Phe Lys

50 55 60

Val Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Arg Asp Ala Cys

65 70 75 80

Asp Pro Ser Val Thr Arg Ser Ala Phe Ala Asp Val Thr Leu Ser Phe

85 90 95

Thr Ser Tyr Ser Thr Asp Glu Glu Arg Ala Leu Ile Arg Thr Glu Leu

100 105 110

Ala Ala Leu Leu Ala Asp Pro Leu Ile Val Asp Ala Ile Asp Asn Leu

115 120 125

Asn Pro Ala Tyr

130

<210>164

<211>130

<212>PRT

<213> phage MS2

<400>164

Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr

1 5 10 15

Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu

20 25 30

Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser

35 40 45

Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu

50 55 60

Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val

65 70 75 80

Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe

85 90 95

Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu

100 105 110

Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly

115 120 125

Ile Tyr

130

<210>165

<211>133

<212>PRT

<213> phage M11

<400>165

Met Ala Lys Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Lys Gly

1 5 10 15

Asp Val Thr Leu Asp Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly

20 25 30

Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg

35 40 45

Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys

50 55 60

Val Gln Val Lys Ile Gln Asn Pro Thr Ser Cys Thr Ala Ser Gly Thr

65 70 75 80

Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr Ser Asp Val Thr Phe Ser

85 90 95

Phe Thr Gln Tyr Ser Thr Val Glu Glu Arg Ala Leu Val Arg Thr Glu

100 105 110

Leu Gln Ala Leu Leu Ala Asp Pro Met Leu Val Asn Ala Ile Asp Asn

115 120 125

Leu Asn Pro Ala Tyr

130

<210>166

<211>133

<212>PRT

<213> phage MX1

<400>166

Met Ala Lys Leu Gln Ala Ile Thr Leu Ser Gly Ile Gly Lys Asn Gly

1 5 10 15

Asp Val Thr Leu Asn Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly

20 25 30

Val Ala Ala Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg

35 40 45

Val Thr Ile Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys

50 55 60

Val Gln Val Lys Ile Gln Asn Pro Thr Ser Cys Thr Ala Ser Gly Thr

65 70 75 80

Cys Asp Pro Ser Val Thr Arg Ser Ala Tyr Ala Asp Val Thr Phe Ser

85 90 95

Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Leu Val Arg Thr Glu

100 105 110

Leu Lys Ala Leu Leu Ala Asp Pro Met Leu Ile Asp Ala Ile Asp Asn

115 120 125

Leu Asn Pro Ala Tyr

130

<210>167

<211>330

<212>PRT

<213> phage NL95

<400>167

Met Ala Lys Leu Asn Lys Val Thr Leu Thr Gly Ile Gly Lys Ala Gly

1 5 10 15

Asn Gln Thr Leu Thr Leu Thr Pro Arg Gly Val Asn Pro Thr Asn Gly

20 25 30

Val Ala Ser Leu Ser Glu Ala Gly Ala Val Pro Ala Leu Glu Lys Arg

35 40 45

Val Thr Val Ser Val Ala Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys

50 55 60

Val Gln Ile Lys Leu Gln Asn Pro Thr Ala Cys Thr Lys Asp Ala Cys

65 70 75 80

Asp Pro Ser Val Thr Arg Ser Gly Ser Arg Asp Val Thr Leu Ser Phe

85 90 95

Thr Ser Tyr Ser Thr Glu Arg Glu Arg Ala Leu Ile Arg Thr Glu Leu

100 105 110

Ala Ala Leu Leu Lys Asp Asp Leu Ile Val Asp Ala Ile Asp Asn Leu

115 120 125

Asn Pro Ala Tyr Trp Ala Ala Leu Leu Ala Ala Ser Pro Gly Gly Gly

130 135 140

Asn Asn Pro Tyr Pro Gly Val Pro Asp Ser Pro Asn Val Lys Pro Pro

145 150 155 160

Gly Gly Thr Gly Thr Tyr Arg Cys Pro Phe Ala Cys Tyr Arg Arg Gly

165 170 175

Glu Leu Ile Thr Glu Ala Lys Asp Gly Ala Cys Ala Leu Tyr Ala Cys

180 185 190

Gly Ser Glu Ala Leu Val Glu Phe Glu Tyr Ala Leu Glu Asp Phe Leu

195 200 205

Gly Asn Glu Phe Trp Arg Asn Trp Asp Gly Arg Leu Ser Lys Tyr Asp

210 215 220

Ile Glu Thr His Arg Arg Cys Arg Gly Asn Gly Tyr Val Asp Leu Asp

225 230 235 240

Ala Ser Val Met Gln Ser Asp Glu Tyr Val Leu Ser Gly Ala Tyr Asp

245 250 255

Val Val Lys Met Gln Pro Pro Gly Thr Phe Asp Ser Pro Arg Tyr Tyr

260 265 270

Leu His Leu Met Asp Gly Ile Tyr Val Asp Leu Ala Glu Val Thr Ala

275 280 285

Tyr Arg Ser Tyr Gly Met Val Ile Gly Phe Trp Thr Asp Ser Lys Ser

290 295 300

Pro Gln Leu Pro Thr Asp Phe Thr Arg Phe Asn Arg His Asn Cys Pro

305 310 315 320

Val Gln Thr Val Ile Val Ile Pro Ser Leu

325 330

<210>168

<211>134

<212>PRT

<213>Apis mellifera

<400>168

Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly His Gly Asn Lys Ser Ser

1 5 10 15

Gly Pro Asn Glu Leu Gly Arg Phe Lys His Thr Asp Ala Cys Cys Arg

20 25 30

Thr His Asp Met Cys Pro Asp Val Met Ser Ala Gly Glu Ser Lys His

35 40 45

Gly Leu Thr Asn Thr Ala Ser His Thr Arg Leu Ser Cys Asp Cys Asp

50 55 60

Asp Lys Phe Tyr Asp Cys Leu Lys Asn Ser Ala Asp Thr Ile Ser Ser

65 70 75 80

Tyr Phe Val Gly Lys Met Tyr Phe Asn Leu Ile Asp Thr Lys Cys Tyr

85 90 95

Lys Leu Glu His Pro Val Thr Gly Cys Gly Glu Arg Thr Glu Gly Arg

100 105 110

Cys Leu His Tyr Thr Val Asp Lys Ser Lys Pro Lys Val Tyr Gln Trp

115 120 125

Phe Asp Leu Arg Lys Tyr

130

<210>169

<211>129

<212>PRT

<213>Apis mellifera

<400>169

Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly His Gly Asn Lys Ser Ser

1 5 10 15

Gly Pro Asn Glu Leu Gly Arg Phe Lys His Thr Asp Ala Cys Cys Arg

20 25 30

Thr His Asp Met Cys Pro Asn Val Met Ser Ala Gly Glu Ser Lys His

35 40 45

Gly Leu Thr Asp Thr Ala Ser Arg Leu Ser Cys Asn Asp Asn Asp Leu

50 55 60

Phe Tyr Lys Asp Ser Ala Asp Thr Ile Ser Ser Tyr Phe Val Gly Lys

65 70 75 80

Met Tyr Phe Asn Leu Ile Asn Thr Lys Cys Tyr Lys Leu Glu His Pro

85 90 95

Val Thr Gly Cys Gly Glu Arg Thr Glu Gly Arg Cys Leu His Tyr Thr

100 105 110

Val Asp Lys Ser Lys Pro Lys Val Tyr Gln Trp Phe Asp Leu Arg Lys

115 120 125

Tyr

<210>170

<211>134

<212>PRT

<213>Apis dorsata

<400>170

Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly His Gly Asn Val Ser Ser

1 5 10 15

Ser Pro Asp Glu Leu Gly Arg Phe Lys His Thr Asp Ser Cys Cys Arg

20 25 30

Ser His Asp Met Cys Pro Asp Val Met Ser Ala Gly Glu Ser Lys His

35 40 45

Gly Leu Thr Asn Thr Ala Ser His Thr Arg Leu Ser Cys Asp Cys Asp

50 55 60

Asp Lys Phe Tyr Asp Cys Leu Lys Asn Ser Ser Asp Thr Ile Ser Ser

65 70 75 80

Tyr Phe Val Gly Glu Met Tyr Phe Asn Ile Leu Asp Thr Lys Cys Tyr

85 90 95

Lys Leu Glu His Pro Val Thr Gly Cys Gly Lys Arg Thr Glu Gly Arg

100 105 110

Cys Leu Asn Tyr Thr Val Asp Lys Ser Lys Pro Lys Val Tyr Gln Trp

115 120 125

Phe Asp Leu Arg Lys Tyr

130

<210>171

<211>134

<212>PRT

<213>Apis cerana

<400>171

Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly His Gly Asn Val Ser Ser

1 5 10 15

Gly Pro Asn Glu Leu Gly Arg Phe Lys His Thr Asp Ala Cys Cys Arg

20 25 30

Thr His Asp Met Cys Pro Asp Val Met Ser Ala Gly Glu Ser Lys His

35 40 45

Gly Leu Thr Asn Thr Ala Ser His Thr Arg Leu Ser Cys Asp Cys Asp

50 55 60

Asp Thr Phe Tyr Asp Cys Leu Lys Asn Ser Gly Glu Lys Ile Ser Ser

65 70 75 80

Tyr Phe Val Gly Lys Met Tyr Phe Asn Leu Ile Asp Thr Lys Cys Tyr

85 90 95

Lys Leu Glu His Pro Val Thr Gly Cys Gly Glu Arg Thr Glu Gly Arg

100 105 110

Cys Leu Arg Tyr Thr Val Asp Lys Ser Lys Pro Lys Val Tyr Gln Trp

115 120 125

Phe Asp Leu Arg Lys Tyr

130

<210>172

<211>136

<212>PRT

<213>Bombus pennsylvanicus

<400>172

Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly Asn Gly Asn Ile Ala Asn

1 5 10 15

Gly Thr Asn Glu Leu Gly Leu Trp Lys Glu Thr Asp Ala Cys Cys Arg

20 25 30

Thr His Asp Met Cys Pro Asp Ile Ile Glu Ala His Gly Ser Lys His

35 40 45

Gly Lau Thr Asn Pro Ala Asp Tyr Thr Arg Leu Asn Cys Glu Cys Asp

50 55 60

Glu Glu Phe Arg His Cys Leu His Asn Ser Gly Asp Ala Val Ser Ala

65 70 75 80

Ala Phe Val Gly Arg Thr Tyr Phe Thr Ile Leu Gly Thr Gln Cys Phe

85 90 95

Arg Leu Asp Tyr Pro Ile Val Lys Cys Lys Val Lys Ser Thr Ile Leu

100 105 110

Arg Glu Cys Lys Glu Tyr Glu Phe Asp Thr Asn Ala Pro Gln Lys Tyr

115 120 125

Gln Trp Phe Asp Val Leu Ser Tyr

130 135

<210>173

<211>142

<212>PRT

<213>Heloderma suspectum

<400>173

Gly Ala Phe Ile Met Pro Gly Thr Leu Trp Cys Gly Ala Gly Asn Ala

1 5 10 15

Ala Ser Asp Tyr Ser Gln Leu Gly Thr Glu Lys Asp Thr Asp Met Cys

20 25 30

Cys Arg Asp His Asp His Cys Ser Asp Thr Met Ala Ala Leu Glu Tyr

35 40 45

Lys His Gly Met Arg Asn Tyr Arg Pro His Thr Val Ser His Cys Asp

50 55 60

Cys Asp Asn Gln Phe Arg Ser Cys Leu Met Asn Val Lys Asp Arg Thr

65 70 75 80

Ala Asp Leu Val Gly Met Thr Tyr Phe Thr Val Leu Lys Ile Ser Cys

85 90 95

Phe Glu Leu Glu Glu Gly Glu Gly Cys Val Asp Asn Asn Phe Ser Gln

100 105 110

Gln Cys Thr Lys Ser Glu Ile Met Pro Val Ala Lys Leu Val Ser Ala

115 120 125

Ala Pro Tyr Gln Ala Gln Ala Glu Thr Gln Ser Gly Glu Gly

130 135 140

<210>174

<211>143

<212>PRT

<213>Heloderma suspectum

<400>174

Gly Ala Phe Ile Met Pro Gly Thr Leu Trp Cys Gly Ala Gly Asn Ala

1 5 10 15

Ala Ser Asp Tyr Ser Gln Leu Gly Thr Glu Lys Asp Thr Asp Met Cys

20 25 30

Cys Arg Asp His Asp His Cys Glu Asn Trp Ile Ser Ala Leu Glu Tyr

35 40 45

Lys His Gly Met Arg Asn Tyr Tyr Pro Ser Thr Ile Ser His Cys Asp

50 55 60

Cys Asp Asn Gln Phe Arg Ser Cys Leu Met Lys Leu Lys Asp Gly Thr

65 70 75 80

Ala Asp Tyr Val Gly Gln Thr Tyr Phe Asn Val Leu Lys Ile Pro Cys

85 90 95

Phe Glu Leu Glu Glu Gly Glu Gly Cys Val Asp Trp Asn Phe Trp Leu

100 105 110

Glu Cys Thr Glu Ser Lys Ile Met Pro Val Ala Lys Leu Val Ser Ala

115 120 125

Ala Pro Tyr Gln Ala Gln Ala Glu Thr Gln Ser Gly Glu Gly Arg

130 135 140

<210>175

<211>142

<212>PRT

<213>Heloderma suspectum

<400>175

Gly Ala Phe Ile Met Pro Gly Thr Leu Trp Cys Gly Ala Gly Asn Ala

1 5 10 15

Ala Ser Asp Tyr Ser Gln Leu Gly Thr Glu Lys Asp Thr Asp Met Cys

20 25 30

Cys Arg Asp His Asp His Cys Glu Asn Trp Ile Ser Ala Leu Glu Tyr

35 40 45

Lys His Gly Met Arg Asn Tyr Tyr Pro Ser Thr Ile Ser His Cys Asp

50 55 60

Cys Asp Asn Gln Phe Arg Ser Cys Leu Met Lys Leu Lys Asp Gly Thr

65 70 75 80

Ala Asp Tyr Val Gly Gln Thr Tyr Phe Asn Val Leu Lys Ile Pro Cys

85 90 95

Phe Glu Leu Glu Glu Gly Glu Gly Cys Val Asp Trp Asn Phe Trp Leu

100 105 110

Glu Cys Thr Glu Ser Lys Ile Met Pro Val Ala Lys Leu Val Ser Ala

115 120 125

Ala Pro Tyr Gln Ala Gln Ala Glu Thr Gln Ser Gly Glu Gly

130 135 140

<210>176

<211>574

<212>PRT

<213> IgE heavy chain

<400>176

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Gln Thr Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys Pro

20 25 30

Gly Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile

35 40 45

Asp Ser Tyr Ile His Trp Ile Arg Gln Ala Pro Gly His Gly Leu Glu

50 55 60

Trp Val Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Pro

65 70 75 80

Arg Phe Gln Gly Arg Val Thr Met Thr Arg Asp Ala Ser Phe Ser Thr

85 90 95

Ala Tyr Met Asp Leu Arg Ser Leu Arg Ser Asp Asp Ser Ala Val Phe

100 105 110

Tyr Cys Ala Lys Ser Asp Pro Phe Trp Ser Asp Tyr Tyr Asn Phe Asp

115 120 125

Tyr Ser Tyr Thr Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

130 135 140

Ser Ser Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg Cys

145 150 155 160

Cys Lys Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys Leu

165 170 175

Ala Thr Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr Gly

180 185 190

Ser Leu Asn Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu Thr Leu

195 200 205

Ser Gly His Tyr Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala Trp

210 215 220

Ala Lys Gln Met Phe Thr Cys Arg Val Ala His Thr Pro Ser Ser Thr

225 230 235 240

Asp Trp Val Asp Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe Thr

245 250 255

Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His

260 265 270

Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro

275 280 285

Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val

290 295 300

Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr

305 310 315 320

Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr

325 330 335

Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr

340 345 350

Lys Lys Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser

355 360 365

Arg Pro Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr

370 375 380

Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr

385 390 395 400

Trp Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu

405 410 415

Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val

420 425 430

Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr

435 440 445

His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser

450 455 460

Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp

465 470 475 480

Pro Gly Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn Phe

485 490 495

Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln Leu

500 505 510

Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly Ser

515 520 525

Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp Glu

530 535 540

Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro

545 550 555 560

Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys

565 570

<210>177

<400>177

000 3

<210>178

<211>13

<212>PRT

<213> IgE peptides

<400>178

Cys Gly Gly Val Asn Leu Thr Trp Ser Arg Ala Ser Gly

1 5 10

<210>179

<211>8

<212>PRT

<213> IgE mimetic

<400>179

Ile Asn His Arg Gly Tyr Trp Val

1 5

<210>180

<211>8

<212>PRT

<213> IgE mimetic

<400>180

Arg Asn His Arg Gly Tyr Trp Val

1 5

<210>181

<211>10

<212>PRT

<213> IgE mimetic

<400>181

Arg Ser Arg Ser Gly Gly Tyr Trp Leu Trp

1 5 10

<210>182

<211>10

<212>PRT

<213> IgE mimetic

<400>182

Val Asn Leu Thr Trp Ser Arg Ala Ser Gly

1 5 10

<210>183

<211>10

<212>PRT

<213> IgE mimetic

<400>183

Val Asn Leu Pro Trp Ser Arg Ala Ser Gly

1 5 10

<210>184

<211>10

<212>PRT

<213> IgE mimetic

<400>184

Val Asn Leu Thr Trp Ser Phe Gly Leu Glu

1 5 10

<210>185

<211>10

<212>PRT

<213> IgE mimetic

<400>185

Val Asn Leu Pro Trp Ser Phe Gly Leu Glu

1 5 10

<210>186

<211>10

<212>PRT

<213> IgE mimetic

<400>186

Val Asn Arg Pro Trp Ser Phe Gly Leu Glu

1 5 10

<210>187

<211>10

<212>PRT

<213> IgE mimetic

<400>187

Val Lys Leu Pro Trp Arg Phe Tyr Gln Val

1 5 10

<210>188

<211>10

<212>PRT

<213> IgE mimetic

<400>188

Val Trp Thr Ala Cys Gly Tyr Gly Arg Met

1 5 10

<210>189

<211>7

<212>PRT

<213> IgE mimetic

<400>189

Gly Thr Val Ser Thr Leu Ser

1 5

<210>190

<211>7

<212>PRT

<213> IgE mimetic

<400>190

Leu Leu Asp Ser Arg Tyr Trp

1 5

<210>191

<211>7

<212>PRT

<213> IgE mimetic

<400>191

Gln Pro Ala His Ser Leu Gly

1 5

<210>192

<211>7

<212>PRT

<213> IgE mimetic

<400>192

Leu Trp Gly Met Gln Gly Arg

1 5

<210>193

<211>15

<212>PRT

<213> IgE mimetic

<400>193

Leu Thr Leu Ser His Pro His Trp Val Leu Asn His Phe Val Ser

1 5 10 15

<210>194

<211>9

<212>PRT

<213> IgE mimetic

<400>194

Ser Met Gly Pro Asp Gln Thr Leu Arg

1 5

<210>195

<211>6

<212>PRT

<213> IgE mimetic

<400>195

Val Asn Leu Thr Trp Ser

1 5

<210>196

<211>56

<212>DNA

<213> oligonucleotide primer

<400>196

tagatgatta cgccaagctt ataatagaaa tagttttttg aaaggaaagc agcatg 56

<210>197

<211>45

<212>DNA

<213> oligonucleotide primer

<400>197

gtcaaaggcc ttgtcgacgt tattccatta cgcccgtcat tttgg 45

<210>198

<211>4623

<212>DNA

<213>pFIMAIC

<400>198

agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 60

tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 120

ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 180

taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 240

tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 300

gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 360

atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 420

ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 480

cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 540

ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 600

aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 660

ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 720

gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 780

ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 840

gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 900

ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 960

tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 1020

cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 1080

tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 1140

atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 1200

tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 1260

tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 1320

ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 1380

cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 1440

ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 1500

gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 1560

tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 1620

gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 1680

ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 1740

tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 1800

ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 1860

tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 1920

attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 1980

tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 2040

ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc 2100

aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt 2160

ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat 2220

gaccatgatt acgccaagct tataatagaa atagtttttt gaaaggaaag cagcatgaaa 2280

attaaaactc tggcaatcgt tgttctgtcg gctctgtccc tcagttctac agcggctctg 2340

gccgctgcca cgacggttaa tggtgggacc gttcacttta aaggggaagt tgttaacgcc 2400

gcttgcgcag ttgatgcagg ctctgttgat caaaccgttc agttaggaca ggttcgtacc 2460

gcatcgctgg cacaggaagg agcaaccagt tctgctgtcg gttttaacat tcagctgaat 2520

gattgcgata ccaatgttgc atctaaagcc gctgttgcct ttttaggtac ggcgattgat 2580

gcgggtcata ccaacgttct ggctctgcag agttcagctg cgggtagcgc aacaaacgtt 2640

ggtgtgcaga tcctggacag aacgggtgct gcgctgacgc tggatggtgc gacatttagt 2700

tcagaaacaa ccctgaataa cggaaccaat accattccgt tccaggcgcg ttattttgca 2760

accggggccg caaccccggg tgctgctaat gcggatgcga ccttcaaggt tcagtatcaa 2820

taacctaccc aggttcaggg acgtcattac gggcagggat gcccaccctt gtgcgataaa 2880

aataacgatg aaaaggaaga gattatttct attagcgtcg ttgctgccaa tgtttgctct 2940

ggccggaaat aaatggaata ccacgttgcc cggcggaaat atgcaatttc agggcgtcat 3000

tattgcggaa acttgccgga ttgaagccgg tgataaacaa atgacggtca atatggggca 3060

aatcagcagt aaccggtttc atgcggttgg ggaagatagc gcaccggtgc cttttgttat 3120

tcatttacgg gaatgtagca cggtggtgag tgaacgtgta ggtgtggcgt ttcacggtgt 3180

cgcggatggt aaaaatccgg atgtgctttc cgtgggagag gggccaggga tagccaccaa 3240

tattggcgta gcgttgtttg atgatgaagg aaacctcgta ccgattaatc gtcctccagc 3300

aaactggaaa cggctttatt caggctctac ttcgctacat ttcatcgcca aatatcgtgc 3360

taccgggcgt cgggttactg gcggcatcgc caatgcccag gcctggttct ctttaaccta 3420

tcagtaattg ttcagcagat aatgtgataa caggaacagg acagtgagta ataaaaacgt 3480

caatgtaagg aaatcgcagg aaataacatt ctgcttgctg gcaggtatcc tgatgttcat 3540

ggcaatgatg gttgccggac gcgctgaagc gggagtggcc ttaggtgcga ctcgcgtaat 3600

ttatccggca gggcaaaaac aagagcaact tgccgtgaca aataatgatg aaaatagtac 3660

ctatttaatt caatcatggg tggaaaatgc cgatggtgta aaggatggtc gttttatcgt 3720

gacgcctcct ctgtttgcga tgaagggaaa aaaagagaat accttacgta ttcttgatgc 3780

aacaaataac caattgccac aggaccggga aagtttattc tggatgaacg ttaaagcgat 3840

tccgtcaatg gataaatcaa aattgactga gaatacgcta cagctcgcaa ttatcagccg 3900

cattaaactg tactatcgcc cggctaaatt agcgttgcca cccgatcagg ccgcagaaaa 3960

attaagattt cgtcgtagcg cgaattctct gacgctgatt aacccgacac cctattacct 4020

gacggtaaca gagttgaatg ccggaacccg ggttcttgaa aatgcattgg tgcctccaat 4080

gggcgaaagc acggttaaat tgccttctga tgcaggaagc aatattactt accgaacaat 4140

aaatgattat ggcgcactta cccccaaaat gacgggcgta atggaataac gtcgactcta 4200

gaggatcccc gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg 4260

gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 4320

cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 4380

gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 4440

tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 4500

ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 4560

gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 4620

gcg 4623

<210>199

<211>42

<212>DNA

<213> oligonucleotide primer

<400>199

aagatcttaa gctaagcttg aattctctga cgctgattaa cc 42

<210>200

<211>41

<212>DNA

<213> oligonucleotide primer

<400>200

acgtaaagca tttctagacc gcggatagta atcgtgctat c 41

<210>201

<211>5681

<212>DNA

<213>pFIMD

<400>201

tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 60

gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 120

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 180

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 240

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 300

gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 360

gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 420

atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 480

caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 540

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 600

accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 660

ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 720

gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 780

acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 840

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 900

ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 960

gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 1020

gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 1080

tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 1140

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 1200

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1260

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1320

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1380

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1440

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1500

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1560

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1620

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1680

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1740

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 1800

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 1860

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 1920

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 1980

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc 2040

aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 2100

gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 2160

ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 2220

caatttcaca caggaaacag ctatgaccat gattacgcca agcttgaatt ctctgacgct 2280

gattaacccg acaccctatt acctgacggt aacagagttg aatgccggaa cccgggttct 2340

tgaaaatgca ttggtgcctc caatgggcga aagcacggtt aaattgcctt ctgatgcagg 2400

aagcaatatt acttaccgaa caataaatga ttatggcgca cttaccccca aaatgacggg 2460

cgtaatggaa taacgcaggg ggaatttttc gcctgaataa aaagaattga ctgccggggt 2520

gattttaagc cggaggaata atgtcatatc tgaatttaag actttaccag cgaaacacac 2580

aatgcttgca tattcgtaag catcgtttgg ctggtttttt tgtccgactc gttgtcgcct 2640

gtgcttttgc cgcacaggca cctttgtcat ctgccgacct ctattttaat ccgcgctttt 2700

tagcggatga tccccaggct gtggccgatt tatcgcgttt tgaaaatggg caagaattac 2760

cgccagggac gtatcgcgtc gatatctatt tgaataatgg ttatatggca acgcgtgatg 2820

tcacatttaa tacgggcgac agtgaacaag ggattgttcc ctgcctgaca cgcgcgcaac 2880

tcgccagtat ggggctgaat acggcttctg tcgccggtat gaatctgctg gcggatgatg 2940

cctgtgtgcc attaaccaca atggtccagg acgctactgc gcatctggat gttggtcagc 3000

agcgactgaa cctgacgatc cctcaggcat ttatgagtaa tcgcgcgcgt ggttatattc 3060

ctcctgagtt atgggatccc ggtattaatg ccggattgct caattataat ttcagcggaa 3120

atagtgtaca gaatcggatt gggggtaaca gccattatgc atatttaaac ctacagagtg 3180

ggttaaatat tggtgcgtgg cgtttacgcg acaataccac ctggagttat aacagtagcg 3240

acagatcatc aggtagcaaa aataaatggc agcatatcaa tacctggctt gagcgagaca 3300

taataccgtt acgttcccgg ctgacgctgg gtgatggtta tactcagggc gatattttcg 3360

atggtattaa ctttcgcggc gcacaattgg cctcagatga caatatgtta cccgatagtc 3420

aaagaggatt tgccccggtg atccacggta ttgctcgtgg tactgcacag gtcactatta 3480

aacaaaatgg gtatgacatt tataatagta cggtgccacc ggggcctttt accatcaacg 3540

atatctatgc cgcaggtaat agtggtgact tgcaggtaac gatcaaagag gctgacggca 3600

gcacgcagat ttttaccgta ccctattcgt cagtcccgct tttgcaacgt gaagggcata 3660

ctcgttattc cattacggca ggagaatacc gtagtggaaa tgcgcagcag gaaaaaaccc 3720

gctttttcca gagtacatta ctccacggcc ttccggctgg ctggacaata tatggtggaa 3780

cgcaactggc ggatcgttat cgtgctttta atttcggtat cgggaaaaac atgggggcac 3840

tgggcgctct gtctgtggat atgacgcagg ctaattccac acttcccgat gacagtcagc 3900

atgacggaca atcggtgcgt tttctctata acaaatcgct caatgaatca ggcacgaata 3960

ttcagttagt gggttaccgt tattcgacca gcggatattt taatttcgct gatacaacat 4020

acagtcgaat gaatggctac aacattgaaa cacaggacgg agttattcag gttaagccga 4080

aattcaccga ctattacaac ctcgcttata acaaacgcgg gaaattacaa ctcaccgtta 4140

ctcagcaact cgggcgcaca tcaacactgt atttgagtgg tagccatcaa acttattggg 4200

gaacgagtaa tgtcgatgag caattccagg ctggattaaa tactgcgttc gaagatatca 4260

actggacgct cagctatagc ctgacgaaaa acgcctggca aaaaggacgg gatcagatgt 4320

tagcgcttaa cgtcaatatt cctttcagcc actggctgcg ttctgacagt aaatctcagt 4380

ggcgacatgc cagtgccagc tacagcatgt cacacgatct caacggtcgg atgaccaatc 4440

tggctggtgt atacggtacg ttgctggaag acaacaacct cagctatagc gtgcaaaccg 4500

gctatgccgg gggaggcgat ggaaatagcg gaagtacagg ctacgccacg ctgaattatc 4560

gcggtggtta cggcaatgcc aatatcggtt acagccatag cgatgatatt aagcagctct 4620

attacggagt cagcggtggg gtactggctc atgccaatgg cgtaacgctg gggcagccgt 4680

taaacgatac ggtggtgctt gttaaagcgc ctggcgcaaa agatgcaaaa gtcgaaaacc 4740

agacgggggt gcgtaccgac tggcgtggtt atgccgtgct gccttatgcc actgaatatc 4800

gggaaaatag agtggcgctg gataccaata ccctggctga taacgtcgat ttagataacg 4860

cggttgctaa cgttgttccc actcgtgggg cgatcgtgcg agcagagttt aaagcgcgcg 4920

ttgggataaa actgctcatg acgctgaccc acaataataa gccgctgccg tttggggcga 4980

tggtgacatc agagagtagc cagagtagcg gcattgttgc ggataatggt caggtttacc 5040

tcagcggaat gcctttagcg ggaaaagttc aggtgaaatg gggagaagag gaaaatgctc 5100

actgtgtcgc caattatcaa ctgccaccag agagtcagca gcagttatta acccagctat 5160

cagctgaatg tcgttaaggg ggcgtgatga gaaacaaacc tttttatctt ctgtgcgctt 5220

ttttgtggct ggcggtgagt cacgctttgg ctgcggatag cacgattact atccgcggtc 5280

tagaggatcc ccgggtaccg agctcgaatt cactggccgt cgttttacaa cgtcgtgact 5340

gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 5400

ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 5460

gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 5520

tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 5580

cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 5640

aagctgtgac cgtctccggg agctgcatgt gtcagaggtt t 5681

<210>202

<211>40

<212>DNA

<213> oligonucleotide primer

<400>202

aattacgtga gcaagcttat gagaaacaaa cctttttatc 40

<210>203

<211>41

<212>DNA

<213> oligonucleotide primer

<400>203

gactaaggcc tttctagatt attgataaac aaaagtcacg c 41

<210>204

<211>4637

<212>DNA

<213>pFIMFGH

<400>204

aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 60

gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 120

aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 180

ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 240

ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 300

agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 360

tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 420

tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 480

ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 540

gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 600

acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 660

tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 720

gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 780

actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 840

aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 900

cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 960

tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 1020

cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 1080

tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 1140

ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 1200

ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 1260

cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 1320

aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 1380

agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 1440

tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 1500

ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 1560

cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 1620

atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 1680

ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 1740

tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 1800

gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 1860

gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 1920

cgcctttgat tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 1980

gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 2040

tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 2100

aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 2160

tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 2220

tgattacgcc aagcttatga gaaacaaacc tttttatctt ctgtgcgctt ttttgtggct 2280

ggcggtgagt cacgctttgg ctgcggatag cacgattact atccgcggct atgtcaggga 2340

taacggctgt agtgtggccg ctgaatcaac caattttact gttgatctga tggaaaacgc 2400

ggcgaagcaa tttaacaaca ttggcgcgac gactcctgtt gttccatttc gtattttgct 2460

gtcaccctgt ggtaatgccg tttctgccgt aaaggttggg tttactggcg ttgcagatag 2520

ccacaatgcc aacctgcttg cacttgaaaa tacggtgtca gcggcttcgg gactgggaat 2580

acagcttctg aatgagcagc aaaatcaaat accccttaat gctccatcgt ccgcgctttc 2640

gtggacgacc ctgacgccgg gtaaaccaaa tacgctgaat ttttacgccc ggctaatggc 2700

gacacaggtg cctgtcactg cggggcatat caatgccacg gctaccttca ctcttgaata 2760

tcagtaactg gagatgctca tgaaatggtg caaacgtggg tatgtattgg cggcaatatt 2820

ggcgctcgca agtgcgacga tacaggcagc cgatgtcacc atcacggtga acggtaaggt 2880

cgtcgccaaa ccgtgtacgg tttccaccac caatgccacg gttgatctcg gcgatcttta 2940

ttctttcagt cttatgtctg ccggggcggc atcggcctgg catgatgttg cgcttgagtt 3000

gactaattgt ccggtgggaa cgtcgagggt cactgccagc ttcagcgggg cagccgacag 3060

taccggatat tataaaaacc aggggaccgc gcaaaacatc cagttagagc tacaggatga 3120

cagtggcaac acattgaata ctggcgcaac caaaacagtt caggtggatg attcctcaca 3180

atcagcgcac ttcccgttac aggtcagagc attgacagta aatggcggag ccactcaggg 3240

aaccattcag gcagtgatta gcatcaccta tacctacagc tgaacccgaa gagatgattg 3300

taatgaaacg agttattacc ctgtttgctg tactgctgat gggctggtcg gtaaatgcct 3360

ggtcattcgc ctgtaaaacc gccaatggta ccgctatccc tattggcggt ggcagcgcca 3420

atgtttatgt aaaccttgcg cccgtcgtga atgtggggca aaacctggtc gtggatcttt 3480

cgacgcaaat cttttgccat aacgattatc cggaaaccat tacagactat gtcacactgc 3540

aacgaggctc ggcttatggc ggcgtgttat ctaatttttc cgggaccgta aaatatagtg 3600

gcagtagcta tccatttcct accaccagcg aaacgccgcg cgttgtttat aattcgagaa 3660

cggataagcc gtggccggtg gcgctttatt tgacgcctgt gagcagtgcg ggcggggtgg 3720

cgattaaagc tggctcatta attgccgtgc ttattttgcg acagaccaac aactataaca 3780

gcgatgattt ccagtttgtg tggaatattt acgccaataa tgatgtggtg gtgcctactg 3840

gcggctgcga tgtttctgct cgtgatgtca ccgttactct gccggactac cctggttcag 3900

tgccaattcc tcttaccgtt tattgtgcga aaagccaaaa cctggggtat tacctctccg 3960

gcacaaccgc agatgcgggc aactcgattt tcaccaatac cgcgtcgttt tcacctgcac 4020

agggcgtcgg cgtacagttg acgcgcaacg gtacgattat tccagcgaat aacacggtat 4080

cgttaggagc agtagggact tcggcggtga gtctgggatt aacggcaaat tatgcacgta 4140

ccggagggca ggtgactgca gggaatgtgc aatcgattat tggcgtgact tttgtttatc 4200

aataatctag aggatccccg ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt 4260

cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 4320

gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 4380

ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 4440

caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 4500

cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 4560

tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 4620

ccgaaacgcg cgagacg 4637

<210>205

<211>9299

<212>DNA

<213>pFIMAICDFGH

<400>205

cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 60

tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 120

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 180

aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 240

tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 300

atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 360

agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 420

tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 480

tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 540

atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 600

ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 660

tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 720

acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 780

ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 840

aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 900

ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 960

cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 1020

gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 1080

actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 1140

agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 1200

cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1260

tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1320

agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 1380

tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 1440

acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 1500

ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 1560

gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 1620

gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 1680

gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 1740

tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 1800

caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 1860

tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 1920

gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 1980

agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 2040

ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 2100

gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 2160

ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 2220

atgaccatga ttacgccaag cttataatag aaatagtttt ttgaaaggaa agcagcatga 2280

aaattaaaac tctggcaatc gttgttctgt cggctctgtc cctcagttct acagcggctc 2340

tggccgctgc cacgacggtt aatggtggga ccgttcactt taaaggggaa gttgttaacg 2400

ccgcttgcgc agttgatgca ggctctgttg atcaaaccgt tcagttagga caggttcgta 2460

ccgcatcgct ggcacaggaa ggagcaacca gttctgctgt cggttttaac attcagctga 2520

atgattgcga taccaatgtt gcatctaaag ccgctgttgc ctttttaggt acggcgattg 2580

atgcgggtca taccaacgtt ctggctctgc agagttcagc tgcgggtagc gcaacaaacg 2640

ttggtgtgca gatcctggac agaacgggtg ctgcgctgac gctggatggt gcgacattta 2700

gttcagaaac aaccctgaat aacggaacca ataccattcc gttccaggcg cgttattttg 2760

caaccggggc cgcaaccccg ggtgctgcta atgcggatgc gaccttcaag gttcagtatc 2820

aataacctac ccaggttcag ggacgtcatt acgggcaggg atgcccaccc ttgtgcgata 2880

aaaataacga tgaaaaggaa gagattattt ctattagcgt cgttgctgcc aatgtttgct 2940

ctggccggaa ataaatggaa taccacgttg cccggcggaa atatgcaatt tcagggcgtc 3000

attattgcgg aaacttgccg gattgaagcc ggtgataaac aaatgacggt caatatgggg 3060

caaatcagca gtaaccggtt tcatgcggtt ggggaagata gcgcaccggt gccttttgtt 3120

attcatttac gggaatgtag cacggtggtg agtgaacgtg taggtgtggc gtttcacggt 3180

gtcgcggatg gtaaaaatcc ggatgtgctt tccgtgggag aggggccagg gatagccacc 3240

aatattggcg tagcgttgtt tgatgatgaa ggaaacctcg taccgattaa tcgtcctcca 3300

gcaaactgga aacggcttta ttcaggctct acttcgctac atttcatcgc caaatatcgt 3360

gctaccgggc gtcgggttac tggcggcatc gccaatgccc aggcctggtt ctctttaacc 3420

tatcagtaat tgttcagcag ataatgtgat aacaggaaca ggacagtgag taataaaaac 3480

gtcaatgtaa ggaaatcgca ggaaataaca ttctgcttgc tggcaggtat cctgatgttc 3540

atggcaatga tggttgccgg acgcgctgaa gcgggagtgg ccttaggtgc gactcgcgta 3600

atttatccgg cagggcaaaa acaagagcaa cttgccgtga caaataatga tgaaaatagt 3660

acctatttaa ttcaatcatg ggtggaaaat gccgatggtg taaaggatgg tcgttttatc 3720

gtgacgcctc ctctgtttgc gatgaaggga aaaaaagaga ataccttacg tattcttgat 3780

gcaacaaata accaattgcc acaggaccgg gaaagtttat tctggatgaa cgttaaagcg 3840

attccgtcaa tggataaatc aaaattgact gagaatacgc tacagctcgc aattatcagc 3900

cgcattaaac tgtactatcg cccggctaaa ttagcgttgc cacccgatca ggccgcagaa 3960

aaattaagat ttcgtcgtag cgcgaattct ctgacgctga ttaacccgac accctattac 4020

ctgacggtaa cagagttgaa tgccggaacc cgggttcttg aaaatgcatt ggtgcctcca 4080

atgggcgaaa gcacggttaa attgccttct gatgcaggaa gcaatattac ttaccgaaca 4140

ataaatgatt atggcgcact tacccccaaa atgacgggcg taatggaata acgcaggggg 4200

aatttttcgc ctgaataaaa agaattgact gccggggtga ttttaagccg gaggaataat 4260

gtcatatctg aatttaagac tttaccagcg aaacacacaa tgcttgcata ttcgtaagca 4320

tcgtttggct ggtttttttg tccgactcgt tgtcgcctgt gcttttgccg cacaggcacc 4380

tttgtcatct gccgacctct attttaatcc gcgcttttta gcggatgatc cccaggctgt 4440

ggccgattta tcgcgttttg aaaatgggca agaattaccg ccagggacgt atcgcgtcga 4500

tatctatttg aataatggtt atatggcaac gcgtgatgtc acatttaata cgggcgacag 4560

tgaacaaggg attgttccct gcctgacacg cgcgcaactc gccagtatgg ggctgaatac 4620

ggcttctgtc gccggtatga atctgctggc ggatgatgcc tgtgtgccat taaccacaat 4680

ggtccaggac gctactgcgc atctggatgt tggtcagcag cgactgaacc tgacgatccc 4740

tcaggcattt atgagtaatc gcgcgcgtgg ttatattcct cctgagttat gggatcccgg 4800

tattaatgcc ggattgctca attataattt cagcggaaat agtgtacaga atcggattgg 4860

gggtaacagc cattatgcat atttaaacct acagagtggg ttaaatattg gtgcgtggcg 4920

tttacgcgac aataccacct ggagttataa cagtagcgac agatcatcag gtagcaaaaa 4980

taaatggcag catatcaata cctggcttga gcgagacata ataccgttac gttcccggct 5040

gacgctgggt gatggttata ctcagggcga tattttcgat ggtattaact ttcgcggcgc 5100

acaattggcc tcagatgaca atatgttacc cgatagtcaa agaggatttg ccccggtgat 5160

ccacggtatt gctcgtggta ctgcacaggt cactattaaa caaaatgggt atgacattta 5220

taatagtacg gtgccaccgg ggccttttac catcaacgat atctatgccg caggtaatag 5280

tggtgacttg caggtaacga tcaaagaggc tgacggcagc acgcagattt ttaccgtacc 5340

ctattcgtca gtcccgcttt tgcaacgtga agggcatact cgttattcca ttacggcagg 5400

agaataccgt agtggaaatg cgcagcagga aaaaacccgc tttttccaga gtacattact 5460

ccacggcctt ccggctggct ggacaatata tggtggaacg caactggcgg atcgttatcg 5520

tgcttttaat ttcggtatcg ggaaaaacat gggggcactg ggcgctctgt ctgtggatat 5580

gacgcaggct aattccacac ttcccgatga cagtcagcat gacggacaat cggtgcgttt 5640

tctctataac aaatcgctca atgaatcagg cacgaatatt cagttagtgg gttaccgtta 5700

ttcgaccagc ggatatttta atttcgctga tacaacatac agtcgaatga atggctacaa 5760

cattgaaaca caggacggag ttattcaggt taagccgaaa ttcaccgact attacaacct 5820

cgcttataac aaacgcggga aattacaact caccgttact cagcaactcg ggcgcacatc 5880

aacactgtat ttgagtggta gccatcaaac ttattgggga acgagtaatg tcgatgagca 5940

attccaggct ggattaaata ctgcgttcga agatatcaac tggacgctca gctatagcct 6000

gacgaaaaac gcctggcaaa aaggacggga tcagatgtta gcgcttaacg tcaatattcc 6060

tttcagccac tggctgcgtt ctgacagtaa atctcagtgg cgacatgcca gtgccagcta 6120

cagcatgtca cacgatctca acggtcggat gaccaatctg gctggtgtat acggtacgtt 6180

gctggaagac aacaacctca gctatagcgt gcaaaccggc tatgccgggg gaggcgatgg 6240

aaatagcgga agtacaggct acgccacgct gaattatcgc ggtggttacg gcaatgccaa 6300

tatcggttac agccatagcg atgatattaa gcagctctat tacggagtca gcggtggggt 6360

actggctcat gccaatggcg taacgctggg gcagccgtta aacgatacgg tggtgcttgt 6420

taaagcgcct ggcgcaaaag atgcaaaagt cgaaaaccag acgggggtgc gtaccgactg 6480

gcgtggttat gccgtgctgc cttatgccac tgaatatcgg gaaaatagag tggcgctgga 6540

taccaatacc ctggctgata acgtcgattt agataacgcg gttgctaacg ttgttcccac 6600

tcgtggggcg atcgtgcgag cagagtttaa agcgcgcgtt gggataaaac tgctcatgac 6660

gctgacccac aataataagc cgctgccgtt tggggcgatg gtgacatcag agagtagcca 6720

gagtagcggc attgttgcgg ataatggtca ggtttacctc agcggaatgc ctttagcggg 6780

aaaagttcag gtgaaatggg gagaagagga aaatgctcac tgtgtcgcca attatcaact 6840

gccaccagag agtcagcagc agttattaac ccagctatca gctgaatgtc gttaaggggg 6900

cgtgatgaga aacaaacctt tttatcttct gtgcgctttt ttgtggctgg cggtgagtca 6960

cgctttggct gcggatagca cgattactat ccgcggctat gtcagggata acggctgtag 7020

tgtggccgct gaatcaacca attttactgt tgatctgatg gaaaacgcgg cgaagcaatt 7080

taacaacatt ggcgcgacga ctcctgttgt tccatttcgt attttgctgt caccctgtgg 7140

taatgccgtt tctgccgtaa aggttgggtt tactggcgtt gcagatagcc acaatgccaa 7200

cctgcttgca cttgaaaata cggtgtcagc ggcttcggga ctgggaatac agcttctgaa 7260

tgagcagcaa aatcaaatac cccttaatgc tccatcgtcc gcgctttcgt ggacgaccct 7320

gacgccgggt aaaccaaata cgctgaattt ttacgcccgg ctaatggcga cacaggtgcc 7380

tgtcactgcg gggcatatca atgccacggc taccttcact cttgaatatc agtaactgga 7440

gatgctcatg aaatggtgca aacgtgggta tgtattggcg gcaatattgg cgctcgcaag 7500

tgcgacgata caggcagccg atgtcaccat cacggtgaac ggtaaggtcg tcgccaaacc 7560

gtgtacggtt tccaccacca atgccacggt tgatctcggc gatctttatt ctttcagtct 7620

tatgtctgcc ggggcggcat cggcctggca tgatgttgcg cttgagttga ctaattgtcc 7680

ggtgggaacg tcgagggtca ctgccagctt cagcggggca gccgacagta ccggatatta 7740

taaaaaccag gggaccgcgc aaaacatcca gttagagcta caggatgaca gtggcaacac 7800

attgaatact ggcgcaacca aaacagttca ggtggatgat tcctcacaat cagcgcactt 7860

cccgttacag gtcagagcat tgacagtaaa tggcggagcc actcagggaa ccattcaggc 7920

agtgattagc atcacctata cctacagctg aacccgaaga gatgattgta atgaaacgag 7980

ttattaccct gtttgctgta ctgctgatgg gctggtcggt aaatgcctgg tcattcgcct 8040

gtaaaaccgc caatggtacc gctatcccta ttggcggtgg cagcgccaat gtttatgtaa 8100

accttgcgcc cgtcgtgaat gtggggcaaa acctggtcgt ggatctttcg acgcaaatct 8160

tttgccataa cgattatccg gaaaccatta cagactatgt cacactgcaa cgaggctcgg 8220

cttatggcgg cgtgttatct aatttttccg ggaccgtaaa atatagtggc agtagctatc 8280

catttcctac caccagcgaa acgccgcgcg ttgtttataa ttcgagaacg gataagccgt 8340

ggccggtggc gctttatttg acgcctgtga gcagtgcggg cggggtggcg attaaagctg 8400

gctcattaat tgccgtgctt attttgcgac agaccaacaa ctataacagc gatgatttcc 8460

agtttgtgtg gaatatttac gccaataatg atgtggtggt gcctactggc ggctgcgatg 8520

tttctgctcg tgatgtcacc gttactctgc cggactaccc tggttcagtg ccaattcctc 8580

ttaccgttta ttgtgcgaaa agccaaaacc tggggtatta cctctccggc acaaccgcag 8640

atgcgggcaa ctcgattttc accaataccg cgtcgttttc acctgcacag ggcgtcggcg 8700

tacagttgac gcgcaacggt acgattattc cagcgaataa cacggtatcg ttaggagcag 8760

tagggacttc ggcggtgagt ctgggattaa cggcaaatta tgcacgtacc ggagggcagg 8820

tgactgcagg gaatgtgcaa tcgattattg gcgtgacttt tgtttatcaa taatctagaa 8880

ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc gtgactggga 8940

aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 9000

taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 9060

atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 9120

gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc 9180

aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 9240

tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcg 9299

<210>206

<211>8464

<212>DNA

<213>pFIMAICDFG

<400>206

cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 60

tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 120

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 180

aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 240

tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 300

atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 360

agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 420

tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 480

tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 540

atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 600

ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 660

tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 720

acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 780

ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 840

aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 900

ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 960

cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 1020

gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 1080

actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 1140

agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 1200

cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 1260

tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 1320

agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 1380

tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 1440

acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 1500

ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 1560

gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 1620

gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 1680

gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 1740

tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 1800

caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 1860

tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 1920

gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 1980

agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 2040

ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 2100

gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 2160

ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 2220

atgaccatga ttacgccaag cttataatag aaatagtttt ttgaaaggaa agcagcatga 2280

aaattaaaac tctggcaatc gttgttctgt cggctctgtc cctcagttct acagcggctc 2340

tggccgctgc cacgacggtt aatggtggga ccgttcactt taaaggggaa gttgttaacg 2400

ccgcttgcgc agttgatgca ggctctgttg atcaaaccgt tcagttagga caggttcgta 2460

ccgcatcgct ggcacaggaa ggagcaacca gttctgctgt cggttttaac attcagctga 2520

atgattgcga taccaatgtt gcatctaaag ccgctgttgc ctttttaggt acggcgattg 2580

atgcgggtca taccaacgtt ctggctctgc agagttcagc tgcgggtagc gcaacaaacg 2640

ttggtgtgca gatcctggac agaacgggtg ctgcgctgac gctggatggt gcgacattta 2700

gttcagaaac aaccctgaat aacggaacca ataccattcc gttccaggcg cgttattttg 2760

caaccggggc cgcaaccccg ggtgctgcta atgcggatgc gaccttcaag gttcagtatc 2820

aataacctac ccaggttcag ggacgtcatt acgggcaggg atgcccaccc ttgtgcgata 2880

aaaataacga tgaaaaggaa gagattattt ctattagcgt cgttgctgcc aatgtttgct 2940

ctggccggaa ataaatggaa taccacgttg cccggcggaa atatgcaatt tcagggcgtc 3000

attattgcgg aaacttgccg gattgaagcc ggtgataaac aaatgacggt caatatgggg 3060

caaatcagca gtaaccggtt tcatgcggtt ggggaagata gcgcaccggt gccttttgtt 3120

attcatttac gggaatgtag cacggtggtg agtgaacgtg taggtgtggc gtttcacggt 3180

gtcgcggatg gtaaaaatcc ggatgtgctt tccgtgggag aggggccagg gatagccacc 3240

aatattggcg tagcgttgtt tgatgatgaa ggaaacctcg taccgattaa tcgtcctcca 3300

gcaaactgga aacggcttta ttcaggctct acttcgctac atttcatcgc caaatatcgt 3360

gctaccgggc gtcgggttac tggcggcatc gccaatgccc aggcctggtt ctctttaacc 3420

tatcagtaat tgttcagcag ataatgtgat aacaggaaca ggacagtgag taataaaaac 3480

gtcaatgtaa ggaaatcgca ggaaataaca ttctgcttgc tggcaggtat cctgatgttc 3540

atggcaatga tggttgccgg acgcgctgaa gcgggagtgg ccttaggtgc gactcgcgta 3600

atttatccgg cagggcaaaa acaagagcaa cttgccgtga caaataatga tgaaaatagt 3660

acctatttaa ttcaatcatg ggtggaaaat gccgatggtg taaaggatgg tcgttttatc 3720

gtgacgcctc ctctgtttgc gatgaaggga aaaaaagaga ataccttacg tattcttgat 3780

gcaacaaata accaattgcc acaggaccgg gaaagtttat tctggatgaa cgttaaagcg 3840

attccgtcaa tggataaatc aaaattgact gagaatacgc tacagctcgc aattatcagc 3900

cgcattaaac tgtactatcg cccggctaaa ttagcgttgc cacccgatca ggccgcagaa 3960

aaattaagat ttcgtcgtag cgcgaattct ctgacgctga ttaacccgac accctattac 4020

ctgacggtaa cagagttgaa tgccggaacc cgggttcttg aaaatgcatt ggtgcctcca 4080

atgggcgaaa gcacggttaa attgccttct gatgcaggaa gcaatattac ttaccgaaca 4140

ataaatgatt atggcgcact tacccccaaa atgacgggcg taatggaata acgcaggggg 4200

aatttttcgc ctgaataaaa agaattgact gccggggtga ttttaagccg gaggaataat 4260

gtcatatctg aatttaagac tttaccagcg aaacacacaa tgcttgcata ttcgtaagca 4320

tcgtttggct ggtttttttg tccgactcgt tgtcgcctgt gcttttgccg cacaggcacc 4380

tttgtcatct gccgacctct attttaatcc gcgcttttta gcggatgatc cccaggctgt 4440

ggccgattta tcgcgttttg aaaatgggca agaattaccg ccagggacgt atcgcgtcga 4500

tatctatttg aataatggtt atatggcaac gcgtgatgtc acatttaata cgggcgacag 4560

tgaacaaggg attgttccct gcctgacacg cgcgcaactc gccagtatgg ggctgaatac 4620

ggcttctgtc gccggtatga atctgctggc ggatgatgcc tgtgtgccat taaccacaat 4680

ggtccaggac gctactgcgc atctggatgt tggtcagcag cgactgaacc tgacgatccc 4740

tcaggcattt atgagtaatc gcgcgcgtgg ttatattcct cctgagttat gggatcccgg 4800

tattaatgcc ggattgctca attataattt cagcggaaat agtgtacaga atcggattgg 4860

gggtaacagc cattatgcat atttaaacct acagagtggg ttaaatattg gtgcgtggcg 4920

tttacgcgac aataccacct ggagttataa cagtagcgac agatcatcag gtagcaaaaa 4980

taaatggcag catatcaata cctggcttga gcgagacata ataccgttac gttcccggct 5040

gacgctgggt gatggttata ctcagggcga tattttcgat ggtattaact ttcgcggcgc 5100

acaattggcc tcagatgaca atatgttacc cgatagtcaa agaggatttg ccccggtgat 5160

ccacggtatt gctcgtggta ctgcacaggt cactattaaa caaaatgggt atgacattta 5220

taatagtacg gtgccaccgg ggccttttac catcaacgat atctatgccg caggtaatag 5280

tggtgacttg caggtaacga tcaaagaggc tgacggcagc acgcagattt ttaccgtacc 5340

ctattcgtca gtcccgcttt tgcaacgtga agggcatact cgttattcca ttacggcagg 5400

agaataccgt agtggaaatg cgcagcagga aaaaacccgc tttttccaga gtacattact 5460

ccacggcctt ccggctggct ggacaatata tggtggaacg caactggcgg atcgttatcg 5520

tgcttttaat ttcggtatcg ggaaaaacat gggggcactg ggcgctctgt ctgtggatat 5580

gacgcaggct aattccacac ttcccgatga cagtcagcat gacggacaat cggtgcgttt 5640

tctctataac aaatcgctca atgaatcagg cacgaatatt cagttagtgg gttaccgtta 5700

ttcgaccagc ggatatttta atttcgctga tacaacatac agtcgaatga atggctacaa 5760

cattgaaaca caggacggag ttattcaggt taagccgaaa ttcaccgact attacaacct 5820

cgcttataac aaacgcggga aattacaact caccgttact cagcaactcg ggcgcacatc 5880

aacactgtat ttgagtggta gccatcaaac ttattgggga acgagtaatg tcgatgagca 5940

attccaggct ggattaaata ctgcgttcga agatatcaac tggacgctca gctatagcct 6000

gacgaaaaac gcctggcaaa aaggacggga tcagatgtta gcgcttaacg tcaatattcc 6060

tttcagccac tggctgcgtt ctgacagtaa atctcagtgg cgacatgcca gtgccagcta 6120

cagcatgtca cacgatctca acggtcggat gaccaatctg gctggtgtat acggtacgtt 6180

gctggaagac aacaacctca gctatagcgt gcaaaccggc tatgccgggg gaggcgatgg 6240

aaatagcgga agtacaggct acgccacgct gaattatcgc ggtggttacg gcaatgccaa 6300

tatcggttac agccatagcg atgatattaa gcagctctat tacggagtca gcggtggggt 6360

actggctcat gccaatggcg taacgctggg gcagccgtta aacgatacgg tggtgcttgt 6420

taaagcgcct ggcgcaaaag atgcaaaagt cgaaaaccag acgggggtgc gtaccgactg 6480

gcgtggttat gccgtgctgc cttatgccac tgaatatcgg gaaaatagag tggcgctgga 6540

taccaatacc ctggctgata acgtcgattt agataacgcg gttgctaacg ttgttcccac 6600

tcgtggggcg atcgtgcgag cagagtttaa agcgcgcgtt gggataaaac tgctcatgac 6660

gctgacccac aataataagc cgctgccgtt tggggcgatg gtgacatcag agagtagcca 6720

gagtagcggc attgttgcgg ataatggtca ggtttacctc agcggaatgc ctttagcggg 6780

aaaagttcag gtgaaatggg gagaagagga aaatgctcac tgtgtcgcca attatcaact 6840

gccaccagag agtcagcagc agttattaac ccagctatca gctgaatgtc gttaaggggg 6900

cgtgatgaga aacaaacctt tttatcttct gtgcgctttt ttgtggctgg cggtgagtca 6960

cgctttggct gcggatagca cgattactat ccgcggctat gtcagggata acggctgtag 7020

tgtggccgct gaatcaacca attttactgt tgatctgatg gaaaacgcgg cgaagcaatt 7080

taacaacatt ggcgcgacga ctcctgttgt tccatttcgt attttgctgt caccctgtgg 7140

taatgccgtt tctgccgtaa aggttgggtt tactggcgtt gcagatagcc acaatgccaa 7200

cctgcttgca cttgaaaata cggtgtcagc ggcttcggga ctgggaatac agcttctgaa 7260

tgagcagcaa aatcaaatac cccttaatgc tccatcgtcc gcgctttcgt ggacgaccct 7320

gacgccgggt aaaccaaata cgctgaattt ttacgcccgg ctaatggcga cacaggtgcc 7380

tgtcactgcg gggcatatca atgccacggc taccttcact cttgaatatc agtaactgga 7440

gatgctcatg aaatggtgca aacgtgggta tgtattggcg gcaatattgg cgctcgcaag 7500

tgcgacgata caggcagccg atgtcaccat cacggtgaac ggtaaggtcg tcgccaaacc 7560

gtgtacggtt tccaccacca atgccacggt tgatctcggc gatctttatt ctttcagtct 7620

tatgtctgcc ggggcggcat cggcctggca tgatgttgcg cttgagttga ctaattgtcc 7680

ggtgggaacg tcgagggtca ctgccagctt cagcggggca gccgacagta ccggatatta 7740

taaaaaccag gggaccgcgc aaaacatcca gttagagcta caggatgaca gtggcaacac 7800

attgaatact ggcgcaacca aaacagttca ggtggatgat tcctcacaat cagcgcactt 7860

cccgttacag gtcagagcat tgacagtaaa tggcggagcc actcagggaa ccattcaggc 7920

agtgattagc atcacctata cctacagctg aacccgaaga gatgattgta atgaaacgag 7980

ttattaccct gtttgctgta ctgctgatgg gctggtcggt aaatgcctgg tcattcgcct 8040

gtaaaaccgc caatggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac 8100

tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 8160

tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 8220

ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 8280

atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac 8340

ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 8400

caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 8460

cgcg 8464

<210>207

<211>13

<212>PRT

<213> Ce3 epitope

<400>207

Cys Gly Gly Val Asn Leu Thr Trp Ser Arg Ala Ser Gly

1 5 10

<210>208

<211>13

<212>PRT

<213> Ce3 analog bit

<400>208

Cys Gly Gly Val Asn Leu Pro Trp Ser Phe Gly Leu Glu

1 5 10

<210>209

<211>9

<212>PRT

<213> bee venom phospholipase A2 cloning vector

<400>209

Ala Ala Ala Ser Gly Gly Cys Gly Gly

1 5

<210>210

<211>145

<212>PRT

<213>PLA₂Fusion proteins

<400>210

Met Ala Ile Ile Tyr Pro Gly Thr Leu Trp Cys Gly His Gly Asn Lys

1 5 10 15

Ser Ser Gly Pro Asn Glu Leu Gly Arg Phe Lys His Thr Asp Ala Cys

20 25 30

Cys Arg Thr Gln Asp Met Cys Pro Asp Val Met Ser Ala Gly Glu Ser

35 40 45

Lys His Gly Leu Thr Asn Thr Ala Ser His Thr Arg Leu Ser Cys Asp

50 55 60

Cys Asp Asp Lys Phe Tyr Asp Cys Leu Lys Asn Ser Ala Asp Thr Ile

65 70 75 80

Ser Ser Tyr Phe Val Gly Lys Met Tyr Phe Asn Leu Ile Asp Thr Lys

85 90 95

Cys Tyr Lys Leu Glu His Pro Val Thr Gly Cys Gly Glu Arg Thr Glu

100 105 110

Gly Arg Cys Leu His Tyr Thr Val Asp Lys Ser Lys Pro Lys Val Tyr

115 120 125

Gln Trp Phe Asp Leu Arg Lys Tyr Ala Ala Ala Ser Gly Gly Cys Gly

130 135 140

Gly

145

<210>211

<211>17

<212>PRT

<213> CE4 simulation bit

<400>211

Gly Glu Phe Cys Ile Asn His Arg Gly Tyr Trp Val Cys Gly Asp Pro

1 5 10 15

Ala

<210>212

<211>27

<212>PRT

<213> synthetic M2 peptide

<400>212

Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys

1 5 10 15

Arg Cys Asn Gly Ser Ser Asp Gly Gly Gly Cys

20 25

<210>213

<211>97

<212>PRT

<213> matrix protein M2

<400>213

Met Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly

1 5 10 15

Cys Arg Cys Asn Gly Ser Ser Asp Pro Leu Ala Ile Ala Ala Asn Ile

20 25 30

Ile Gly Ile Leu His Leu Ile Leu Trp Ile Leu Asp Arg Leu Phe Phe

35 40 45

Lys Cys Ile Tyr Arg Arg Phe Lys Tyr Gly Leu Lys Gly Gly Pro Ser

50 55 60

Thr Glu Gly Val Pro Lys Ser Met Arg Glu Glu Tyr Arg Lys Glu Gln

65 70 75 80

Gln Ser Ala Val Asp Ala Asp Asp Gly His Phe Val Ser Ile Glu Leu

85 90 95

Glu

<210>214

<211>42

<212>DNA

<213> oligonucleotide

<400>214

taaccgaatt caggaggtaa aaacatatgg ctatcatcta cc 42

<210>215

<211>129

<212>PRT

<213> phage f2

<400>215

Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asn Asp Gly Gly Thr Gly

1 5 10 15

Asn Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu Trp

20 25 30

Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val

35 40 45

Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val

50 55 60

Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val Ala

65 70 75 80

Ala Trp Arg Ser Tyr Leu Asn Leu Glu Leu Thr Ile Pro Ile Phe Ala

85 90 95

Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu

100 105 110

Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly Ile

115 120 125

Tyr

<210>216

<211>17

<212>PRT

<213> Ring analog bit

<400>216

Gly Glu Phe Cys Ile Asn His Arg Gly Tyr Trp Val Cys Gly Asp Pro

1 5 10 15

Ala

<210>217

<211>329

<212>PRT

<213> bacteriophage Q-beta

<400>217

Met Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly

1 5 10 15

Lys Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly

20 25 30

Val Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg

35 40 45

Val Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys

50 55 60

Val Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser

65 70 75 80

Cys Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser

85 90 95

Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu

100 105 110

Leu Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln

115 120 125

Leu Asn Pro Ala Tyr Trp Thr Leu Leu Ile Ala Gly Gly Gly Ser Gly

130 135 140

Ser Lys Pro Asp Pro Val Ile Pro Asp Pro Pro Ile Asp Pro Pro Pro

145 150 155 160

Gly Thr Gly Lys Tyr Thr Cys Pro Phe Ala Ile Trp Ser Leu Glu Glu

165 170 175

Val Tyr Glu Pro Pro Thr Lys Asn Arg Pro Trp Pro Ile Tyr Asn Ala

180 185 190

Val Glu Leu Gln Pro Arg Glu Phe Asp Val Ala Leu Lys Asp Leu Leu

195 200 205

Gly Asn Thr Lys Trp Arg Asp Trp Asp Ser Arg Leu Ser Tyr Thr Thr

210 215 220

Phe Arg Gly Cys Arg Gly Asn Gly Tyr Ile Asp Leu Asp Ala Thr Tyr

225 230 235 240

Leu Ala Thr Asp Gln Ala Met Arg Asp Gln Lys Tyr Asp Ile Arg Glu

245 250 255

Gly Lys Lys Pro Gly Ala Phe Gly Asn Ile Glu Arg Phe Ile Tyr Leu

260 265 270

Lys Ser Ile Asn Ala Tyr Cys Ser Leu Ser Asp Ile Ala Ala Tyr His

275 280 285

Ala Asp Gly Val Ile Val Gly Phe Trp Arg Asp Pro Ser Ser Gly Gly

290 295 300

Ala Ile Pro Phe Asp Phe Thr Lys Phe Asp Lys Thr Lys Cys Pro Ile

305 310 315 320

Gln Ala Val Ile Val Val Pro Arg Ala

325

<210>218

<211>770

<212>PRT

<213> amyloid beta protein (Homo sapiens)

<400>218

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg

1 5 10 15

Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro

20 25 30

Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln

35 40 45

Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp

50 55 60

Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu

65 70 75 80

Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn

85 90 95

Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val

100 105 110

Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu

115 120 125

Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys

130 135 140

Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu

145 150 155 160

Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile

165 170 175

Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu

180 185 190

Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val

195 200 205

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys

210 215 220

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu

225 230 235 240

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu

245 250 255

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile

260 265 270

Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg

275 280 285

Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile

290 295 300

Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe

305 310 315 320

Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr

325 330 335

Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr

340 345 350

Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala

355 360 365

Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp

370 375 380

Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala

385 390 395 400

Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala

405 410 415

Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile

420 425 430

Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn

435 440 445

Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met

450 455 460

Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu

465 470 475 480

Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys

485 490 495

Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe

500 505 510

Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser

515 520 525

Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser

530 535 540

Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp

545 550 555 560

Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val

565 570 575

Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala

580 585 590

Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro

595 600 605

Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe

610 615 620

Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val

625 630 635 640

Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser

645 650 655

Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp

660 665 670

Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu

675 680 685

Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly

690 695 700

Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu

705 710 715 720

Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val

725 730 735

Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met

740 745 750

Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met

755 760 765

Gln Asn

770

<210>219

<211>82

<212>PRT

<213> amyloid beta peptide precursor (Homo Sapiens)

<400>219

Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys

1 5 10 15

Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln

20 25 30

Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile

35 40 45

Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Ile Ile

50 55 60

Thr Leu Val Met Leu Lys Lys Gln Tyr Thr Ser Asn His His Gly Val

65 70 75 80

Val Glu

<210>220

<211>42

<212>PRT

<213> amyloid beta peptide

<400>220

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

221：

RANKL _ human: TrEMBL: o14788: extracellular domains

YFRAQMDPNRIS EDGTHCIYRI LRLHENADFQ DTTLESQDTK LTPDSCRRIK QAFQGAVQKE

LQHIVGSQHI RAEKAMVDGS WLDLAKRSKL EAQPFAHLTI NATDIPSGSH KVSLSSWYHD

RGWAKISNMT FSNGKLIVNQ DGFYYLYANI CFRHHETSGD LATEYLQLMV YVTKTSIKIP

SSHTLMKGGS TKYWSGNSEF HFYSINVGGF FKLRSGEEIS IEVSNPSLLD PDQDATYFGA FKVRDID

222：

RANKL _ human: splicing isoform TrEMBL: o14788

MDPNPISEDG THCIYRILRL HENADFQDTT LESQDTKLIP DSCRRIKQAF QEAVQKELQH

IVGSQHIRAE KAMVDGSWLD LAKRSKLEAQ PEAHLTINAT DIPSGSHKVS LSSWYHDRGW

AKISNMTFSN GKLIVNQDGF YYLYANICFR HHETSGDLAT EYLQLMVYVT KTSIKIPSSH

TLMKGGSTKY WSGNSEFHFY SINVGGFFKL RSGEEISIEV SNPSLLDPDQ DATYFGAFKV

RDID

223：

RANKL _ mouse: TrEMBL: o35235: extracellular domains

YFRAQMDPNRI SEDSTHCFYR ILRLHENAGL QDSTLESEDT LPDSCRRMKQV AFQEAVQKEL

QHIVGPQRFS GAPAMMEGSW LDVAQRGKPE AQPFAHLTIN AASIPSHSHK VTLSSWYHDR

GWAKISNMTL SNGKLRVNQD GFYYLYANIC FRHHETSGSV PTDYLQLMVY VVKTSIKTPS

SHNLMKGGST KNWSGNSEFH FYSINVGGFF KLRAGEEISI QVSNPSLLDP DQDATYFGAF KVQDID

224：

RANKL _ mouse splice isoform: TrEMBL: q9JJK8

MKQAFQGAVQ KELQHIVGPQ RFSGAPAMME GSWLDVAQRG KPEAQPFAHL TINAASIPSG

SHKVTLSSWY HDRGWAKISN MTLSNGKLRV NQDGFYYLYA NICFRHHETS GSVPTDYLQL

MVYVVKTSIK IPSSHNLMKG GSTKNWSGNS EFHFYSINVG GFFKLRAGEE ISIQVSNPSL

LDPDQDATYF GAFKVQDID

225：

MIF_rat：SwissProt

PMFIVNTNVP RASVPEGFLS ELTQQLAQAT GKPAQYIAVH VVPDQLMTFS GTSDPCALCS

LHSIGKIGGA QNRNYSKLLC GLLSDRLHIS PDRVYINYYD MNAANVVGWNG STFA

226：

MIF_mouse：SwissProt

PMFIVNTNVP RASVPEGFLS ELTQQLAQAT GKPAQYIAVH VVPDQLMTFS GTNDPCALCS

LHSIGKIGGA QNRNYSKLLC GLLSDRLHIS PDRVYINYYD MNAANVGWNG STFA

227：

MIF_human：SwissProt

PMFIVNTNVP RASVPDGFLS ELTQQLAQAT GKPPQYIAVH VVPDQLMAFG GSSEPCALCS

LHSIGKIGGA QNRSYSKLLC GLLAERLRIS PDRVYINYYD MNAANVGWNN STFA

228：

Human IL-17

Accession number: AAC50341

1 mtpgktslvs lllllsleai vkagitiprn pgcpnsedkn fprtvmvnln ihurntntnp

61 krssdyynrs tspwnlhrne dperypsviw eakcrhlgci nadgnvdyhm nsvpiqqeil

121 vlrrepphcp nsfrlekilv svgctcvtpi vhhva

229：

Mouse IL-17

Accession number: AAA37490

1 mspgrassvs lmlllllsla atvkaaaiip qssacpntea kdflqnvkvn lkvfnslgak

61 vssrrpsdyl nrstspwtlh rnedpdryps viweaqcrhq rcvnaegkld hhmnsvliqq

121 eilvlkrepe scpftfrvek mlvgvgctcv asivrqaa

230：

Human IL-13 (precursor)

MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSG

CSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

231：

Human IL-13 (after processing)

GPVPPSTALR ELIEELVNIT QNQKAPLCNG SMVWSINLTA

GMYCAALESL INVSGCSAIE KTQRMLSGFC PHKVSAGQFS SLHVRDTKIE VAQFVKDLLL

HLKKLFREGR FN

232：

Mouse IL-13 (after processing)

GPVPRSVSLPLTLKELIEELSNITQDQTPLCNGSMVWSVDLAAGGFCVALDSLTNISNCNAIYRTQRILHGLCNR

KAPTTVSSLPDTKIEVAHFITKLLSYTKQLFRHGPF

233：

Human IL-5 (precursor)

MRMLLHLSLL ALGAAYVYAI PTEIPTSALV KETLALLSTH RTLLIANETL RIPVPVHKNH

QLCTEEIFQG IGTLESQTVQ GGTVERLFKN LSLIKKYIDG QKKKCGEERR RVNQFLDYLQEFLGVMNTEW

IIES

234：

Human IL-5 (after processing)

I PTEIPTSALV KETLALLSTH RTLLIANETL RIPVPVHKNH

QLCTEEIFQG IGTLESQTVQ GGTVERLFKN LSLIKKYIDG QKKKCGEERR RVNQFLDYLQ

EFLGVMNTEW IIES

235：

Mouse IL-5 (after processing)

MEIPMSTVVKETLTQLSAHRALLTSNETMRLPVPTHKNHQLCIGEIFQGLDILKNQTVRGGTVEMLFQNLSLIKK

YIDRQKEKCGEERRRTRQFLDYLQEFLGVMSTEWAMEG

236：

CCL21 Swissprot: SY21_ human: sequence after cleavage of the signal peptide:

SDGGAQD CCLKYSQRKI PAKVVRSYRK QEPSLGCSIP AILFLPRKRS QAELCADPKE LWVQQLMQHL

DQPPAPGKQS PGCRKNRGTS KSGKKGKGSK GCKRTEQTQF SRG

237：

CCL21 Swissprot: SY21_ mouse: sequence after cleavage of the signal peptide:

SDGGGQD CCLKYSQKKI PYSIVRGYRK QEPSLGCPIP AILFSPRKHS KPELCANPEE GWVQNLMRRL

DQPPAPGKQS PGCRKNRGTS KSGKKGKGSK GCKRTEQTQP SRG

238：

swissprot: SDF1_ human: sequence after cleavage of the signal peptide:

DGKPVSLSYRC PCRFFESHVA RANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQ

EYLEKALNKR FKM

239：

swissprot: SDF1_ mouse: sequence after cleavage of the signal peptide:

DGKPVSLSYRC PCRFFESHIA RANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQ

EYLEKALNK

240：

BLC sequence: human: accession number: NP-006410

Amino acids 1-22 are signal peptides.

MKFISTSLLL MLLVSSLSPV QGVLEVYYTS LRCRCVQESS VFIPRRFIDR IQILPRGNGC

PRKEIIVWKK NKSIVCVDPQ AEWIQRMMEV LRKRSSSTLP VPVFKRKIP

241：

BLC sequence: mice: accession number: NP _061354

Amino acids 1-21 are signal peptides.

MRLSTATLLL LLASCLSPGH GILEAHYTNL KCRCSGVIST VVGLNIIDRIQVTPPGNGCP

KTEVVIWTKM KKVICVNPRA KWLQRLLRHV QSKSLSSTPQ APVSKRRAA

242：

Human eotaxin-1

1-23 are signal peptides

1 mkvsaallwl lliaaafspq glagpasvpt tccfnlanrk iplqrlesyr ritsgkcpqk

61 avifktklak dicadpkkkw vqdsmkyldq ksptpkp

243：

Human eotaxin-2

1-26 are signal peptides

1 maglmtivts llflgvcahh iiptgsvvip spccmffvsk ripenrwsy qlssrstclk

61 agvifttkkg qqfcgdpkqe wvqrymknld akqkkaspra ravavkgpvq rypgnqttc

244：

Human eotaxin-3

1-23 are signal peptides

1 mmglslasav llasllslhl gtatrgsdis ktccfqyshk plpwtwvrsy eftsnscsqr

61 avifttkrgk kvcthprkkw vqkyisllkt pkql

245：

Mouse eotaxin-1

1-23 are signal peptides

1 mqsstallfl lltvtsftsq vlahpgsipt sccfimtskk ipntllksyk ritnnrctlk

61 aivfktrlgk eicadpkkkw vqdatkhldq klqtpkp

246：

Mouse eotaxin-2

1-25 are signal peptides

1 magsativag llllvacacc ifpidsvtip sscctsfisk kipenrvvsy qlangsicpk

61 agvifitkkg hkictdpkll wvqrhiqk1d akknqpskga kavrtkfavq rrrgnstev

247：

M-CSF sequence: human: this construct will be an N-terminal fragment consisting of residues 33-181 or 33-185,

corresponding to the soluble form of the receptor.

Accession number: NP-000748

MTAPGAAGRC PPTTWLGSLL LLVCLLASRS ITEEVSEYCS HMIGSGHLQS LQRLIDSQME

TSCQITFEFV DQEQLKDPVC YLKKAFLLVQ DIMEDTMRFR DNTPNAIAIV QLQELSLRLK

SCFTKDYEEH DKACVRTFYE TPLQLLEKVK NVFNETKNLL DKDWNIFSKN CNNSFAECSS

QDVVTKPDCN CLYPKAIPSS DPASVSPHQP LAPSMAPVAG LTWEDSEGTE GSSLLPGEQP

LHTVDPGSAK QRPPRSTCQS FEPPETPVVK DSTIGGSPQP RPSVGAFNPG MEDILDSAMG

TNWVPEEASG EASEIPVPQG TELSPSRPGG GSMQTEPARP SNFLSASSPL PASAKGQQPA

DVTGTALPRV GPVRPTGQDW NHTPQKTDHP SALLRDPPEP GSPRISSPRP QGLSNPSTLS

AQPQLSRSHS SGSVLPLGEL EGRRSTRDRR SPAEPEGGPA SEGAARPLPR FNSVPLTDTH

ERQSEGSSSP QLQESVFHLL VPSVILVILA VGGLLFYRWR RRSHQEPQRA DSPLEQPEGS

PLTQDDRQVE LPV

248：

M-CSF mouse sequence: the mature sequence starts with amino acid 33. Accession number:

NP_031804

MTARGAAGRC PSSTWLGSRL LLVCLLMSRS IAKEVSEHCS HMIGNGHLKV LQQLIDSQME

TSCQIAFEFV DQEQLDDPVC YLKKAFFLVQ DIIDETMRFK DNTPNANATE RLQELSNNLN

SCFTKDYEEQ NKACVRTFHE TPLQLLEKIK NFFNETKNLL EKDWNIFTKN CNNSFAKCSS

RDVVTKPDCN CLYPKATPSS DPASASPHQP PAPSMAPLAG LAWDDSQRTE GSSLLPSELP

LRIEDPGSAK QRPPRSTCQT LESTEQPNHG DRLTEDSQPH PSAGGPVPGV EDILESSLGT

NWVLEEASGE ASEGFLTQEA KFSPSTPVGG SIQAETDRPR ALSASPFPKS TEDQKPVDIT

DRPLTEVNPM RPIGQTQNNT PEKTDGTSTL REDHQEPGSP HIATPNPQRV SNSATPVAQL

LLPKSHSWGI VLPLGELEGK RSTRDRRSPA ELEGGSASEG AARPVARFNS IPLTDTGHVE

QHEGSSDPQI PESVFHLLVP GIILVLLTVG GLLFYKWKWR SHRDPQTLDS SVGRPEDSSL

TQDEDRQVEL PV

249：

the sequence of human resistin: a precursor.

MKALCLLLLPVLGLLVSSKTLCSMEEAINERIQEVAGSLIFRAISSIGLECQSVTSRGDLATCPRGFAVTGCTCG

SACGSWDVRAETTCHCQCAGMDWTGARCCRVQP

250：

Sequence of mouse resistin: a precursor.

MKNLSFPLLFLFFLVPELLGSSMPLCPIDEAIDKKIKQDFNSLFPNAIKNIGLNCWTVSSRGKLASCPEGTAVLSCSC

GSACGSWDIREEKVCHCQCARIDWTAARCCKLQVAS

251：

Lymphotoxin- β:

swissprot: TNFC _ human: sequence of the extracellular domain:

QD QGGLVTETAD PGAQAQQGLG FQKLPEEEPE TDLSPGLPAA HLIGAPLKGQ GLGWETTKEQ

AFLTSGTQFS DAEGLALPQD GLYYLYCLVG YRGRAPPGGG DPQGRSVTLR SSLYRAGGAY GPGTPELLLE

GAETVTPVLD PARRQGYGPL WYTSVGFGGL VQLRRGERVY VN

252：

lymphotoxin- β:

swissprot: TNFC _ mouse: sequence of the extracellular domain:

QD QGRRVEKIIG SGAQAQKRLD DSKPSCILPS PSSLSETPDP RLHPQRSNAS RNLASTSQGP

VAQSSREASA WMTILSPAAD STPDPGVQQL PKGEPETDLN PELPAAHLIG AWMSGQGLSW

EASQEEAFLR SGAQFSPTHG LALPQDGVYY LYCHVGYRGR TPPAGRSRAR SLTLRSALYR

AGGAYGRGSP ELLLEGAETV TPVVDPIGYG SLWYTSVGFG GLAQLRSGER VYVNISHPDM

VDYRRGKTFF GAVMVG

253：

RNA-phage PP 7:

msktivlsvg eatrtlteiq stadrqifee kvgplvgrlr ltaslrqnga ktayrvnlkl

dqadvvdcst svcgelpkvr ytqvwshdvt ivansteasr kslydltksl vatsqvedlv

vnlvplgr

254：

RNA-phage SPA1 protein:

aklnqvtls kigkngdqtl tltprgvnpt ngvaslseag avpalekrvt vsvaqpsrnr

knfkvqiklq nptactrdac dpsvtrsafa dvtlsftsys tdeeralirt elaalladpl

ivdaidnlnp aywaallvas sgggdnpsdp dvpvvpdvkp pdgtgrykcp facyrlgsiy

evgkegspdi yergdevsvt fdyaledflg ntnwrnwdqr lsdydianrr rcrgngyidl

datamqsddf vlsgrygvrk vkfpgafgsi kyllniqgda wldlsevtay rsygmvigfw

tdskspqlpt dftqfnsanc pvqtviiips 1

255：

“Qβ240”：

AKLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

256：

“Qβ243”：

AKLETVTLGLIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

257：

“Qβ250”：

ARLETVTLGNIGRDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

258：

“Qβ259”：

ARLETVTLGNIGKDGRQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

259：

“Qβ251”：

AKLETVTLGNIGKDGRQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

260：

PH19(SEQ ID NO：260)

TAAGTCCTCTGCCACGTACC

261：

PH20(SEQ ID NO：261)

TGGAAACCACGCTCACTTCC

262：

PH21(SEQ ID NO：262)

CGGGATCCGGGATGAAGAACCTTTCATTTC

263：

PH22(SEQ ID NO：263)

GCCTCTAGAGAGGAAGCGACCTGCAGCTTAC

264：

PH29(SEQ ID NO：264)

CTAGCGGGAGGGGGTGGATGTGGGGACGACTACAAGGATGACGACA

265：

PH30(SEQ ID NO：265)

AGCTTGTCGTCATCCTTGTAGTCGTCCCCACATCCACCCCCTCCCG

266：

PH31(SEQ ID NO：266)

AGCTTACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAGG

267：

PH32(SEQ ID NO：267)

CGGCTTCAGGTGCTGGGCACGGTGGGCATGTGTGAGTA

268：

PH35(SEQ ID NO：268)

CTAGCGGGAGGGGGTGGATGTGGGATCGAAGGTCGCA

269：

PH36(SEQ ID NO：269)

AGCTTGCGACCTTCGATCCCACATCCACCCCCTCCCG

270：

PH37(SEQ ID NO：270)

CGGGATCCAGCAGCTGGGCTCGAGGTGCTAGCTTTGTTTAAAC

271：

PH38(SEQ ID NO：271)

GATCGTTTAAACAAACAAAGCTAGCACCTCGAGCCCAGCTGCTGGATCCCGGTAC

272：

PH39(SEQ ID NO：272)

CTAGCGGGAGGGGGTGGATGTGGGGACGATGACGACA

273：

PH40(SEQ ID NO：273)

AGCTTGTCGTCATCGTCCCCACATCCACCCCCTCCCG

274：

PH41(SEQ ID NO：274)

CATGGAGACAGACACACTCCTGCTATGGGT

275：

PH42(SEQ ID NO：275)

GCAGTACCCATAGCAGGAGTGTGTCTGTCTCCATGGTAC

276：

PH43(SEQ ID NO：276)

ACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACGCG

277：

PH44(SEQ ID NO：277)

GATCCGCGTCACCAGTGGAACCTGGAACCCAGAGCA

278：

SU7(SEQ ID NO：278)

AGCTTGCGGATCCAGGATATCGGCTCGAGGTTCTAGAGTG

279：

SU8(SEQ ID NO：279)

GGCCCACTCTAGAACCTCGAGCCGATATCCTGGATCCGCA

280：

resistin-C-Xa:

SSMPLCPIDEAIDKKIKQDFNSLFPNAIKNIGLNCWTVSSRGKLASCPEGTAVLSCSCG

SACGSWDIREEKVCHCQCARIDWTAARCCKLQVASSLAGGGGCGIEGR

281：

resistin-C-EK

SSMPLCPIDEAIDKKIKQDFNSLFPNAIKNIGLNCWTVSSRGKLASCPEGTAVLSCSCG

SACGSWDIREEKVCHCQCARIDWTAARCCKLQVASSLAGGGGCGDDDD

282：

resistin-GCG:

SSMPLCPIDEAIDKIKQDFNSLFPNAIKNIGLNCWTVSSRGKLASCPEGTAVLSCSCG

SACGSWDIREEKVCHCQCARIDWTAARCCKLQVASSLAGGGGCG

283：

pCep-Xa-Fc: (complete sequence)

1 GCCCCGCCGC CGGACGAACT AAACCTGACT ACGGCATCTC TGCCCCTTCT TCGCTGGTAC GAGGAGCGCT

71 TTTGTTTTGT ATTCGGGGCA GTGCATGTAA TCCCTTCAGT TGGTTGGTAC AACTTGCCAA CTGGGCCCTG

141 TTCCACATGT GACACGGGGG GGGACCAAAC ACAAAGGGGT TCTCTGACTG TAGTTGACAT CCTTATAAAT

211 GGATGTGCAC ATTTGCCAAC ACTGAGTGGC TTTCATCCTG GAGCAGACTT TGCATGCTGT GGACTGCAAC

281 ACAACATTGC CTTTATGTGT AACTCTTGGC TGAAGCTCTT ACACCAATGC TGGGGGACAT GTACCTCCCA

351 GGGGCCCAGG AAGACTACGG GAGGCTACAC CAACGTCAAT CAGAGGGGCC TGTGTAGCTA CCGATAAGCG

421 GACCCTCAAG AGGGCATTAG CAATAGTGTT TATAAGGCCC CCTTGTTAAC CCTAAACGGG TAGCATATGC

491 TTCCCGGGTA GTAGTATATA CTATCCAGAC TAACCCTAAT TCAATAGCAT ATGTTACCCA ACGGGAAGCA

561 TATGCTATCG AATTAGGGTT AGTAAAAGGG TCCTAAGGAA CAGCGATATC TCCCACCCCA TGAGCTGTCA

631 CGGTTTTATT TACATGGGGT CAGGATTCCA CGAGGGTAGT GAACCATTTT AGTCACAAGG GCAGTGGCTG

701 AAGATCAAGG AGCGGGCAGT GAACTCTCCT GAATCTTCGC CTGCTTCTTC ATTCTCCTTC GTTTAGCTAA

771 TAGAATAACT GCTGAGTTGT GAACAGTAAG GTGTATGTGA GGTGCTCGAA AACAAGGTTT CAGGTGACGC

841 CCCCAGAATA AAATTTGGAC GGGGGGTTCA GTGGTGGCAT TGTGCTATGA CACCAATATA ACCCTCACAA

911 ACCCCTTGGG CAATAAATAC TAGTGTAGGA ATGAAACATT CTGAATATCT TTAACAATAG AAATCCATGG

981 GGTGGGGACA AGCCGTAAAG ACTGGATGTC CATCTCACAC GAATTTATGG CTATGGGCAA CACATAATCC

1051 TAGTGCAATA TGATACTGGG GTTATTAAGA TGTGTCCCAG GCAGGGACCA AGACAGGTGA ACCATGTTGT

1121 TACACTCTAT TTGTAACAAG GGGAAAGAGA GTGGACGCCG ACAGCAGCGG ACTCCACTGG TTGTCTCTAA

1191 CACCCCCGAA AATTAAACGG GGCTCCACGC CAATGGGGCC CATAAACAAA GACAAGTGGC CACTCTTTTT

1261 TTTGAAATTG TGGAGTGGGG GCACGCGTCA GCCCCCACAC GCCGCCCTGC GGTTTTGGAC TGTAAAATAA

1331 GGGTGTAATA ACTTGGCTGA TTGTAACCCC GCTAACCACT GCGGTCAAAC CACTTGCCCA CAAAACCACT

1401 AATGGCACCC CGGGGAATAC CTGCATAAGT AGGTGGGCGG GCCAAGATAG GGGCGCGATT GCTGCGATCT

1471 GGAGGACAAA TTACACACAC TTGCGCCTGA GCGCCAAGCA CAGGGTTGTT GGTCCTCATA TTCACGAGGT

1541 CGCTGAGAGC ACGGTGGGCT AATGTTGCCA TGGGTAGCAT ATACTACCCA AATATCTGGA TAGCATATGC

1611 TATCCTAATC TATATCTGGG TAGCATAGGC TATCCTAATC TATATCTGGG TAGCATATGC TATCCTAATC

1681 TATATCTGGG TAGTATATGC TATCCTAATT TATATCTGGG TAGCATAGGC TATCCTAATC TATATCTGGG

1751 TAGCATATGC TATCCTAATC TATATCTGGG TAGTATATGC TATCCTAATC TGTATCCGGG TAGCATATGC

1821 TATCCTAATA GAGATTAGGG TAGTATATGC TATCCTAATT TATATCTGGG TAGCATATAC TACCCAAATA

1891 TCTGGATAGC ATATGCTATC CTAATCTATA TCTGGGTAGC ATATGCTATC CTAATCTATA TCTGGGTAGC

1961 ATAGGCTATC CTAATCTATA TCTGGGTAGC ATATGCTATC CTAATCTATA TCTGGGTAGT ATATGCTATC

2031 CTAATTTATA TCTGGGTAGC ATAGGCTATC CTAATCTATA TCTGGGTAGC ATATGCTATC CTAATCTATA

2101 TCTGGGTAGT ATATGCTATC CTAATCTGTA TCCGGGTAGC ATATGCTATC CTCATGCATA TACAGTCAGC

2171 ATATGATACC CAGTAGTAGA GTGGGAGTGC TATCCTTTGC ATATGCCGCC ACCTCCCAAG GGGGCGTGAA

2241 TTTTCGCTGC TTGTCCTTTT CCTGCATGCT GGTTGCTCCC ATTCTTAGGT GAATTTAAGG AGGCCAGGCT

2311 AAAGCCGTCG CATGTCTGAT TGCTCACCAG GTAAATGTCG CTAATGTTTT CCAACGCGAG AAGGTGTTGA

2381 GCGCGGAGCT GAGTGACGTG ACAACATGGG TATGCCCAAT TGCCCCATGT TGGGAGGACG AAAATGGTGA

2451 CAAGACAGAT GGCCAGAAAT ACACCAACAG CACGCATGAT GTCTACTGGG GATTTATTCT TTAGTGCGGG

2521 GGAATACACG GCTTTTAATA CGATTGAGGG CGTCTCCTAA CAAGTTACAT CACTCCTGCC CTTCCTCACC

2591 CTCATCTCCA TCACCTCCTT CATCTCCGTC ATCTCCGTCA TCACCCTCCG CGGCAGCCCC TTCCACCATA

2661 GGTGGAAACC AGGGAGGCAA ATCTACTCCA TCGTCAAAGC TGCACACAGT CACCCTGATA TTGCAGGTAG

2731 GAGCGGGCTT TGTCATAACA AGGTCCTTAA TCGCATCCTT CAAAACCTCA GCAAATATAT GAGTTTGTAA

2801 AAAGACCATG AAATAACAGA CAATGGACTC CCTTAGCGGG CCAGGTTGTG GGCCGGGTCC AGGGGCCATT

2871 CCAAAGGGGA GACGACTCAA TGGTGTAAGA CGACATTGTG GAATAGCAAG GGCAGTTCCT CGCCTTAGGT

2941 TGTAAAGGGA GGTCTTACTA CCTCCATATA CGAACACACC GGCGACCCAA GTTCCTTCGT CGGTAGTCCT

3011 TTCTACGTGA CTCCTAGCCA GGAGAGCTCT TAAACCTTCT GCAATGTTCT CAAATTTCGG GTTGGAACCT

3081 CCTTGACCAC GATGCTTTCC AAACCACCCT CCTTTTTTGC GCCTGCCTCC ATCACCCTGA CCCCGGGGTC

3151 CAGTGCTTGG GCCTTCTCCT GGGTCATCTG CGGGGCCCTG CTCTATCGCT CCCGGGGGCA CGTCAGGCTC

3221 ACCATCTGGG CCACCTTCTT GGTGGTATTC AAAATAATCG GCTTCCCCTA CAGGGTGGAA AAATGGCCTT

3291 CTACCTGGAG GGGGCCTGCG CGGTGGAGAC CCGGATGATG ATGACTGACT ACTGGGACTC CTGGGCCTCT

3361 TTTCTCCACG TCCACGACCT CTCCCCCTGG CTCTTTCACG ACTTCCCCCC CTGGCTCTTT CACGTCCTCT

3431 ACCCCGGCGG CCTCCACTAC CTCCTCGACC CCGGCCTCCA CTACCTCCTC GACCCCGGCC TCCACTGCCT

3501 CCTCGACCCC GGCCTCCACC TCCTGCTCCT GCCCCTCCTG CTCCTGCCCC TCCTCCTGCT CCTGCCCCTC

3571 CTGCCCCTCC TGCTCCTGCC CCTCCTGCCC CTCCTGCTCC TGCCCCTCCT GCCCCTCCTG CTCCTGCCCC

3641 TCCTGCCCCT CCTCCTGCTC CTGCCCCTCC TGCCCCTCCT CCTGCTCCTG CCCCTCCTGC CCCTCCTGCT

3711 CCTGCCCCTC CTGCCCCTCC TGCTCCTGCC CCTCCTGCCC CTCCTGCTCC TGCCCCTCCT GCTCCTGCCC

3781 CTCCTGCTCC TGCCCCTCCT GCTCCTGCCC CTCCTGCCCC TCCTGCCCCT CCTCCTGCTC CTGCCCCTCC

3851 TGCTCCTGCC CCTCCTGCCC CTCCTGCCCC TCCTGCTCCT GCCCCTCCTC CTGCTCCTGC CCCTCCTGCC

3921 CCTCCTGCCC CTCCTCCTGC TCCTGCCCCT CCTGCCCCTC CTCCTGCTCC TGCCCCTCCT CCTGCTCCTG

3991 CCCCTCCTGC CCCTCCTGCC CCTCCTCCTG CTCCTGCCCC TCCTGCCCCT CCTCCTGCTC CTGCCCCTCC

4061 TCCTGCTCCT GCCCCTCCTG CCCCTCCTGC CCCTCCTCCT GCTCCTGCCC CTCCTCCTGC TCCTGCCCCT

4131 CCTGCCCCTC CTGCCCCTCC TGCCCCTCCT CCTGCTCCTG CCCCTCCTCC TGCTCCTGCC CCTCCTGCTC

4201 CTGCCCCTCC CGCTCCTGCT CCTGCTCCTG TTCCACCGTG GGTCCCTTTG CAGCCAATGC AACTTGGACG

4271 TTTTTGGGGT CTCCGGACAC CATCTCTATG TCTTGGCCCT GATCCTGAGC CGCCCGGGGC TCCTGGTCTT

4341 CCGCCTCCTC GTCCTCGTCC TCTTCCCCGT CCTCGTCCAT GGTTATCACC CCCTCTTCTT TGAGGTCCAC

4411 TGCCGCCGGA GCCTTCTGGT CCAGATGTGT CTCCCTTCTC TCCTAGGCCA TTTCCAGGTC CTGTACCTGG

4481 CCCCTCGTCA GACATGATTC ACACTAAAAG AGATCAATAG ACATCTTTAT TAGACGACGC TCAGTGAATA

4551 CAGGGAGTGC AGACTCCTGC CCCCTCCAAC AGCCCCCCCA CCCTCATCCC CTTCATGGTC GCTGTCAGAC

4621 AGATCCAGGT CTGAAAATTC CCCATCCTCC GAACCATCCT CGTCCTCATC ACCAATTACT CGCAGCCCGG

4691 AAAACTCCCG CTGAACATCC TCAAGATTTG CGTCCTGAGC CTCAAGCCAG GCCTCAAATT CCTCGTCCCC

4761 CTTTTTGCTG GACGGTAGGG ATGGGGATTC TCGGGACCCC TCCTCTTCCT CTTCAAGGTC ACCAGACAGA

4831 GATGCTACTG GGGCAACGGA AGAAAAGCTG GGTGCGGCCT GTGAGGATCA GCTTATCGAT GATAAGCTGT

4901 CAAACATGAG AATTCTTGAA GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA

4971 ATAATGGTTT CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT

5041 TCTAAATACA TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA

5111 AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT

5181 GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA CGAGTGGGTT

5251 ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT

5321 GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTGTTGACG CCGGGCAAGA GCAACTCGGT

5391 CGCCGCATAC ACTATTCTCA GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG

5461 GCATGACAGT AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT

5531 GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT

5601 GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG CCTGCAGCAA

5671 TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT TACTCTAGCT TCCCGGCAAC AATTAATAGA

5741 CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT

5811 GATAAATCTG GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT

5881 CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA

5951 GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT TTAGATTGAT

6021 TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA TAATCTCATG ACCAAAATCC

6091 CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC

6161 TTTTTTTCTG CGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG

6231 GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTCC

6301 TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT

6371 AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC AAGACGATAG

6441 TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA

6511 CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC

6581 GGACAGGTAT CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC

6651 TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG

6721 GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGCGCCGC

6791 GTGCGGCTGC TGGAGATGGC GGACGCGATG GATATGTTCT GCCAAGGGTT GGTTTGCGCA TTCACAGTTC

6861 TCCGCAAGAA TTGATTGGCT CCAATTCTTG GAGTGGTGAA TCCGTTAGCG AGGCCATCCA GCCTCGCGTC

6931 GAACTAGATG ATCCGCTGTG GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA

7001 GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG

7071 CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG

7141 CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT TATGCAGAGG

7211 CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG AGGAGGCTTT TTTGGAGGGT GACCGCCACG

7281 ACCGGTGCCG CCACCATCCC CTGACCCACG CCCCTGACCC CTCACAAGGA GACGACCTTC CATGACCGAG

7351 TACAAGCCCA CGGTGCGCCT CGCCACCCGC GACGACGTCC CCCGGGCCGT ACGCACCCTC GCCGCCGCGT

7421 TCGCCGACTA CCCCGCCACG CGCCACACCG TCGACCCCGA CCGCCACATC GAACGCGTCA CCGAGCTGCA

7491 AGAACTCTTC CTCACGCGCG TCGGGCTCGA CATCGGCAAG GTGTGGGTCG CGGACGACGG CGCCGCGGTG

7561 GCGGTCTGGA CCACGCCGGA GAGCGTCGAA GCGGGGGCGG TGTTCGCCGA GATCGGCCCG CGCATGGCCG

7631 AGTTGAGCGG TTCCCGGCTG GCCGCGCAGC AACAGATGGA AGGCCTCCTG GCGCCGCACC GGCCCAAGGA

7701 GCCCGCGTGG TTCCTGGCCA CCGTCGGCGT CTCGCCCGAC CACCAGGGCA AGGGTCTGGG CAGCGCCGTC

7771 GTGCTCCCCG GAGTGGAGGC GGCCGAGCGC GCCGGGGTGC CCGCCTTCCT GGAGACCTCC GCGCCCCGCA

7841 ACCTCCCCTT CTACGAGCGG CTCGGCTTCA CCGTCACCGC CGACGTCGAG TGCCCGAAGG ACCGCGCGAC

7911 CTGGTGCATG ACCCGCAAGC CCGGTGCCTG ACGCCCGCCC CACGACCCGC AGCGCCCGAC CGAAAGGAGC

7981 GCACGACCCG GTCCGACGGC GGCCCACGGG TCCCAGGGGG GTCGACCTCG AAACTTGTTT ATTGCAGCTT

8051 ATAATGGTTA CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAG

8121 TTGTGGTTTG TCCAAACTCA TCAATGTATC TTATCATGTC TGGATCGATC CGAACCCCTT CCTCGACCAA

8191 TTCTCATGTT TGACAGCTTA TCATCGCAGA TCCGGGCAAC GTTGTTGCAT TGCTGCAGGC GCAGAACTGG

8261 TAGGTATGGA AGATCTATAC ATTGAATCAA TATTGGCAAT TAGCCATATT AGTCATTGGT TATATAGCAT

8331 AAATCAATAT TGGCTATTGG CCATTGCATA CGTTGTATCT ATATCATAAT ATGTACATTT ATATTGGCTC

8401 ATGTCCAATA TGACCGCCAT GTTGACATTG ATTATTGACT AGTTATTAAT AGTAATCAAT TACGGGGTCA

8471 TTAGTTCATA GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC

8541 CCAACGACCC CCGCCCATTG ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA

8611 TTGACGTCAA TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA

8681 AGTCCGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC ATGACCTTAC

8751 GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC ATGGTGATGC GGTTTTGGCA

8821 GTACACCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA TTTCCAAGTC TCCACCCCAT TGACGTCAAT

8891 GGGAGTTTGT TTTGGCACCA AAATCAACGG GACTTTCCAA AATGTCGTAA TAACCCCGCC CCGTTGACGC

8961 AAATGGGCGG TAGGCGTGTA CGGTGGGAGG TCTATATAAG CAGAGCTCGT TTAGTGAACC GTCAGATCTC

9031 TAGAAGCTGG GTACCGGGAT CCAGCAGCTG GGCTCGAGGT GCTAGCGGGA GGGGGTGGAT GTGGGATCGA

9101 AGGTCGCAAG CTTACTCACA CATGCCCACC GTGCCCAGCA CCTGAAGCCG AGGGGGCACC GTCAGTCTTC

9171 CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA GGTCACATGC GTGGTGGTGG

9241 ACGTGAGCCA CGAAGACCCT GAGGTCAAGT TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA

9311 GACAAAGCCG CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT CCTGCACCAG

9381 GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA ACAAAGCCCT CCCAGCCTCC ATCGAGAAAA

9451 CCATCTCCAA AGCCAAAGGG CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGATGAGCT

9521 GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTATCCCA GCGACATCGC CGTGGAGTGG

9591 GAGAGCAATG GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGTT GGACTCCGAC GGCTCCTTCT

9661 TCCTCTACAG CAAGCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT

9731 GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGTAAATG ACTCGAGGCC

9801 CGAACAAAAA CTCATCTCAG AAGAGGATCT GAATAGCGCC GTCGACCATC ATCATCATCA TCATTGAGTT

9871 TNAACGATCC AGACATGATA AGATACATTG ATGAGTTTGG ACAAACCACA ACTAGAATGC AGTGAAAAAA

9941 ATGCTTTATT TGTGAAATTT GTGATGCTAT TGCTTTATTT GTAACCATTA TAAGCTGCAA TAAACAAGTT

10011 AACAACAACA ATTGCATTCA TTTTATGTTT CAGGTTCAGG GGGAGGTGGG GAGGTTTTTT AAAGCAAGTA

10081 AAACCTCTAC AAATGTGGTA TGGCTGATTA TGATCCGGCT GCCTCGCGCG TTTCGGTGAT GACGGTGAAA

10151 ACCTCTGACA CATGCAGCTC CCGGAGACGG TCACAGCTTG TCTGTAAGCG GATGCCGGGA GCAGACAAGC

10221 CCGTCAGGGC GCGTCAGCGG GTGTTGGCGG GTGTCGGGGC GCAGCCATGA CCGGTCGACT CTAGA

5’LT·：(SEQ ID NO：284)

5’-CTT GGT GCC GCA GGA TCA G-3’

285：

3’LT·：(SEQ ID NO：285)

5’-CAG ATG GCT GTC ACC CCA C-3’

286：

5’LT·long-NheI：(SEQ ID NO：286)

5’-GCC CGC TAG CCT GCG GTG GTC AGG ATC AGG GAC GTC G-3’

287：

5’LT·short-NheI：(SEQ ID NO：287)

5’-GCC CGC TAG CCT GCG GTG GTT CTC CAG CTG CGG ATT C-3’

288：

3’LT·stop-NotI：(SEQ ID NO：288)

5’-CAA TGA CTG CGG CCG CTT ACC CCA CCA TCA CCG-3’

289：

GST-EK-C-LT·_49-306：SEQ ID NO：289

APLVMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQ

SMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCH

KTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATF

GGGDHPPKASMTGGQQMGRDLYDDDDKLACGGQDQGRRVEKllGSGAQAQKRLDDSKPSCILPSPSSL

SETPDPRLHPQRSNASRNLASTSQGPVAQSSREASAWMTILSPAADSTPDPGVQQLPKGEPETDLNPEL

PAAHLIGAWMSGQGLSWEASQEEAFLRSGAQFSPTHGLALPQDGVYYLYCHVGYRGRTPPAGRSRARS

LTLRSALYRAGGAYGRGSPELLLEGAETVTPVVDPIGYGSLWYTSVGFGGLAQLRSGERVYVNISHPDMV

DYRRGKTFFGAVMVG

290：

GST-EK-C-LT·_126-306：SEQ ID NO：290

APLVMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQ

SMAllRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCH

KTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATF

GGGDHPPKASMTGGQQMGRDLYDDDDKLACGGSPAADSTPDPGVQQLPKGEPETDLNPELPAAHLlGA

WMSGQGLSWEASQEEAFLRSGAQFSPTHGLALPQDGVYYLYCHVGYRGRTPPAGRSRARSLTLRSALY

RAGGAYGRGSPELLLEGAETVTPVVDPIGYGSLWYTSVGFGGLAQLRSGERVYVNISHPDMVDYRRGKT

FFGAVMVG

291：

his-myc-EK-C-LT·_49-306：SEQ ID NO：291

APLVHHHHHHGPLVDVASNEQKLISEEDLASMTGGQQMGRDLYDDDDKLACGGQDQGRRVEKIIGSGAQ

AQKRLDDSKPSCILPSPSSLSETPDPRLHPQRSNASRNLASTSQGPVAQSSREASAWMTILSPAADSTPDPGV

QQLPKGEPETDLNPELPAAHLIGAWMSGQGLSWEASQEEAFLRSGAQFSPTHGLALPQDGVYYLYCHVGY

RGRTPPAGRSRARSLTLRSALYRAGGAYGRGSPELLLEGAETVTPVVDPIGYGSLWYTSVGFGGLAQLRSG

ERVYVNISHPDMVDYRRGKTFFGAVMVG

292：

his-myc-EK-C-LT·_126-306：SEQ ID NO：292

APLVHHHHHHGPLVDVASNEQKLISEEDLASMTGGQQMGRDLYDDDDKLACGGSPAADSTPDPGVQQLP

KGEPETDLNPELPAAHLIGAWMSGQGLSWEASQEEAFLRSGAQFSPTHGLALPQDGVYYLYCHVGYRGRT

PPAGRSRARSLTLRSALYRAGGAYGRGSPELLLEGAETVTPVVDPIGYGSLWYTSVGFGGLAQLRSGERVY

VNISHPDMVDYRRGKTFFGAVMVG

293：

Primer MCS-1F

5’-TAT GGA TCC GGC TAG CGC TCG AGG GTT TAA ACG GCG GCC GCA T-3’(SEQ ID NO：293)

294：

Primer MCS-1R

5’-TCG AAT GCG GCC GCC GTT TAA ACC CTC GAG CGC TAG CCG GAT CCA-3’(SEQ ID NO：294)

295：

Bamhis6-EK-Nhe-F

5’-GAT CCA CAC CAC CAC CAC CAC CAC GGT TCT GGT GAC GAC GAT GAC AAA GCG CTA GCC C-3’(SEQ ID NO：295)

296：

Bamhis6-EK-Nhe-R

5’-TCG AGG GCT AGC GCT TTG TCA TCG TCG TCA CCA GAA CCG TGG TGG TGG TGG TGG TGT G-3’

(SEQ ID NO：296)

297：

oligo 1F-C-Glycine-linker

5’-TCG AGG GTG GTG GTG GTG GTT GCG GTT AAT AAG TTT AAA CGC-3’(SEQ ID NO：297)

298：

oligo 1R-C-Glycine-linker

5’-GGC CGC GTT TAA ACT TAT TAA CCG CAA CCA CCA CCA CCA CCC-3’(SEQ ID NO：298)

299：

oligo 1F-C-gamma 1-linker

5’-TCG AGG ATA AAA CCC ACA CCT CTC CGC CGT GTG GTT AAT AAG TTT AAA CGC-3’(SEQ IDNO：299)

300：

oligo lR-C-gamma 1-linker

5’-GGC CGC GTT TAA ACT TAT TAA CCA CAC GGC GGA GAG GTG TGG GTT TTA TCC-3’(SEQ IDNO：300)

301：

oligo1 FA-C-gamma 3-linker

5’-TCG AGC CGA AAC CGT CTA CCC CGC CGG GTT CTT CTG-3’(SEQ ID NO：301)

302：

oligo1 RA-C-gamma 3-linker

5’-CAC CAC CAG AAG AAC CCG GCG GGG TAG ACG GTT TCG GC-3’(SEQ ID NO：302)

303：

oligo2 FB-C-gamma 3-linker

5’-GTG GTG CTC CGG GTG GTT GCG GTT AAT AAG TTT AAA CGC-3’(SEQ ID NO：303)

304：

oligo2 RB-C-gamma 3-linker

5’-GGC CGC GTT TAA ACT TAT TAA CCG CAA CCA CCC GGA G-3’(SEQ ID NO：304)

305：

rMIF-F

5’-GGA ATT CCA TAT GCC TAT GTTCAT CGT GAA CAC-3’(SEQ ID NO：305)

306：

rMIF-Xho-R

5’-CCC GCT CGA GAG CGA AGG TGG AAC CGT TC-3’(SEQ ID NO：306)

307：

rMIF-C1：

MPMFIVNTNVPRASVPEGFLSELTQQLAQATGKPAQYIAVHVVPDQLMTFSGTSDPCALCSLHSIGKIGGAQ

NRNYSKLLCGLLSDRLHISPDRVYINYYDMNAANVGWNGSTFALEGGGGGCG(SEQ ID NO：307)

308：

rMIFF-C2

MPMFIVNTNVPRASVPEGFLSELTQQLAQATGKPAQYIAVHVVPDQLMTFSGTSDPCALCSLHSIGKIGGAQ

NRNYSKILLCGLLSDRLHISPDRVYINYYDMNAANVGWNGSTFALEDKTHTSPPCG(SEQ ID NO：308)

309：

rMIF-C3

MPMFIVNTNVPRASVPEGFLSELTQQLAQATGKPAQYIAVHVVPDQLMTFSGTSDPCALCSLHSIGKIGGAQ

NRNYSKLLCGLLSDRLHISPDRVYINYYDMNAANVGWNGSTFALEPKPSTPPGSSGGAPGGCG(SEQ IDNO：309)

310：

met-human-MIF-C1

MPMFIVNTNVP RASVPDGFLS ELTQQLAQAT GKPPQYIAVH VVPDQLMAFG GSSEPCALCS

LHSIHKIGGA QNRSYSKLLC GLLAERLRIS PDRVYINYYD MNAANVGWNN STFALEGGGGGCG

311：

human-MIF-C1 (SEQ ID NO: 311)

PMFIVNTNVP RASVPDGFLS ELTQQLAQAT GKPPQYIAVH VVPDQLMAFG GSSEPCALCS

LHSIGKIGGA QNRSYSKLLC GLLAERLRIS PDRVYINYYD MNAANVGWNN STFALEGGGGGCG

312：

met-human-MIF-C2 (SEQ ID NO: 312)

MPMFIVNTNVP RASVPDGFLS ELTQQLAQAT GKPPQYIAVH VVPDQLMAFG GSSEPCALCS

LHSIGKIGGA QNRSYSKLLC GLLAERLRIS PDRVYINYYD MNAANVGWNN STFALEDKTHTSPPCG

313：

human-MIF-C2 (SEQ ID NO: 313)

PMFIVNTNVP RASVPDGFLS ELTQQLAQAT GKPPQYIAVH VVPDQLMAFG GSSEPCALCS

LHSIGKIGGA QNRSYSKLLC GLLAERLRIS PDRVYINYYD MNAANVGWNN STFALEDKTHTSPPCG

314：

met-human-MIF-C3 (SEQ ID NO: 314)

MPMFIVNTNVP RASVPDGFLS ELTQQLAQAT GKPPQYIAVH VVPDQLMAFG GSSEPCALCS

LHSIGKIGGA QNRSYSKLLCGLLAERLRISPDRVYINYYD MNAANVGWNN STFALEPKPSTPPGSSGGAPGGCG

315：

human-MIF-C3 (SEQ ID NO: 315)

PMFIVNTNVP RASVPDGFLS ELTQQLAQAT GKPPQYIAVH VVPDQLMAFG GSSEPCALCS

LHSIGKIGGA QNRSYSKLLCGLLAERLRISPDRVYINYYD MNAANVGWNN STFALEPKPSTPPGSSGGAPGGCG

316：

RANKL-UP：

5’CTGCCAGGGGCCCGGGTGCGGCGGTGGCCATCATCACCACCATCACCAGCGCTTCTCAGGAG-3’

317：

RANKL-DOWN：

5’-CCGCTCGAGTTAGTCTATGTCCTGAACTTTGAAAG-3’

318 and 319:

protein sequence of GST-PS-C-RANKL (SEQ ID NO: 318; capital letters)

cDNA sequence of GST-PS-C-RANKL (SEQ ID NO: 319; lower case letters)

1 M S P I L G Y W K I K G L V Q P T R L L L E Y L E

1 atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaa

26 E K Y E E H L Y E R D E G D K W R N K K F E L G L

76 gaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttg

51 E F P N L P Y Y I D G D V K L T Q S M A I I R Y I

151 gagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatata

76 A D K H N M L G G C P K E R A E I S M L E G A V L

226 gctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttg

101 D I R Y G V S R I A Y S K D F E T L K V D F L S K

301 gatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaag

126 L P E M L K M F E D R L C H K T Y L N G D H V T H

376 ctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccat

151 P D F M L Y D A L D V V L Y M D P M C L D A F P K

451 cctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaa

176 L V C F K K R I E A I P Q I D K Y L K S S K Y I A

526 ttagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagca

201 W P L Q G W Q A T F G G G D H P P K S D L E V L F

601 tggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatctggaagttctgttc

226 Q G P G C G G G H H H H H M Q R F S G A P A M M E

676 cagGGGCCCGGGTGCGGCGGTGGCCATCATCACCACCATCACCAGCGCTTCTCAGGAGCTCCAGCTATGATGGAA

251 G S W L D V A Q R G K P E A Q P F A H L T I N A A

751 GGCTCATGGTTGGATGTGGCCCAGCGAGGCAAGCCTGAGGCCCAGCCATTTGCACACCTCACCATCAATGCTGCC

276 S I P S G S H K V T L S S W Y H D R G W A K J S N

826 AGCATCCCATCGGGTTCCCATAAAGTCACTCTGTCCTCTTGGTACCACGATCGAGGCTGGGCCAAGATCTCTAAC

301 M T L S N G K L R V N Q D G F Y Y L Y A N I C F R

901 ATGACGTTAAGCAACGGAAAACTAAGGGTTAACCAAGATGGCTTCTATTACCTGTACGCCAACATTTGCTTTCGG

326 H H E T S G S V P T D Y L Q L M V Y V V K T S I K

976 CATCATGAAACATCGGGAAGCGTACCTACAGACTATCTTCAGCTGATGGTGTATGTCGTTAAAACCAGCATCAAA

351 I P S S H N L M K G G S T K N W S G N S E F H F Y

1051 ATCCCAAGTTCTCATAACCTGATGAAAGGAGGGAGCACGAAAAACTGGTCGGGCAATTCTGAATTCCACTTTTAT

376 S I N V G G F F K L R A G E E I S I Q V S N P S L

1126 TCCATAAATGTTGGGGGATTTTTCAAGCTCCGAGCTGGTGAAGAAATTAGCATTCAGGTGTCCAACCCTTCCCTG

401 L D P D Q D A T Y F G A F K V Q D I D

1201 CTGGATCCGGATCAAGATGCGACGTACTTTGGGGCTTTCAAAGTTCAGGACATAGACTAACTCGAGCGG

320：

human-C-RANKL

GCGGGQHIRAEKAMVDGSWLDLAKRSKLEAQPFAHLTINATDIPSGSHKVSLSSWYHDRGWAKISNMTFSNGKLI

VNQDGFYYLYANICFRHHETSGDLATEYLQLMVYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFK

LRSGEEISIEVSNPSLLDPDQDATYFGAFKVRDID

321：

Primer 5' PrP-BamHI

5’-CGG GAT CCC ACC ATG GTG GGG GGC CTT GG-3’(SEQ ID NO：321)

322：

Primer 3' PrP-NheI

5’-CTA GCT AGC CTG GAT CTT CTC CCG-3’(SEQ ID NO：322)

323：

mPrP_t-EK-Fc protein sequence

MVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHDCVNJTIKQHT

VTTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYDGRSRLAGGGGCGDDDDKLTHTCPPCPAPEA

EGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

K

324：

mPrP_t

MVGGLGGYMLGSAMSRPMIHFGNDWEDRYYRENMYRYPNQVYYRPVDQYSNQNNFVHDCVNITIKQHT

VTTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYDGRSRLAGGGGCGDDDDK

325：

Human resistin-C-Xa: (SEQ ID NO: 325)

SSKTLCSMEEAINERIQEVAGSLIFRAISSIGLECQSVTSRGDL

ATCPRGFAVTGCTCGSACGSWDVRAETTCHCQCAGMDWTGARCCRVQPGGGGCG

IEGR

326：

Human resistin-C-EK: (SEQ ID NO: 326)

SSKTLCSMEEAINERIQEVAGSLIFRAISSIGLECQSVTSRGDL

ATCPRGFAVTGCTCGSACGSWDVRAETTCHCQCAGMDWTGARCCRVQPGGGGCG

DDDDK

327：

Human resistin-C: (SEQ ID NO: 327)

SSKTLCSMEEAINERIQEVAGSLIFDAISSIGLECQSVTSRGDL

ATCPRGFAVTGCTCGSACGSWDVRAETTCHCQCAGMDWTGARCCRVQPGGGGCG

328：

Mouse C-IL-13-F: (SEQ ID NO: 328)

ADPGCGGGGGLAGPVPRSVSLPLTLKELIEELSNITQDQTPLCNGSMVWSVDLAAGGFCVALDSLTNISNCN

AIYRTQRILHGLCNRKAPTTVSSLPDTKIEVAHFITKLLSYTKQLFRHGPFLEVLAIEGR

329：

Mouse C-IL-13-S: (SEQ ID NO: 329)

LACGGGGGGPVPRSVSLPLTLKELIEELSNITQDQTPLCNGSMVWSVDLAAGGFCVALDSLTNISNCNAI

YRTQRILHGLCNRKAPTTVSSLPDTKIEVAHFITKLLSYTKQLFRHGPF

330：

Human C-IL-13-F: (SEQ ID NO: 330)

ADPGCGGGGGLAGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCS

AIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNLEVLAIEGR

331：

Human C-IL-13-S: (SEQ ID NO: 331)

LACGGGGGGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQR

MLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN

332：

Mouse C-IL-5-E: (SEQ ID NO: 332)

ALVGCGGPKPSTPPGSSGGAPASMEIPMSTVVKETLTQLSAHRALLTSNETMRLPVPTHKNHQLCIGEIFQG

LDILKNQTVRGGTVEMLFQNLSLIKKYIDRQKEKCGEERRRTRQFLDYLQEFLGVMSTEWAMEG

333：

Mouse C-IL-5-F: (SEQ ID NO: 333)

ADPGCGGGGGLAMEIPMSTVVKETLTQLSAHRALTSNETMRLPVPTHKNHQLCIGEIFQGLDILKNQTVR

GGTVEMLFQNLSLIKKYIDRQKEKCGEERRRTRQFLDYLQEFLGVMSTEWAMEGLEVLAIEGR

334：

Mouse C-IL-5-S: (SEQ ID NO: 334)

LACGGGGGMEIPMSTVVKETLTQLSAHRALLTSNETMRLPVPTHKNHQLCIGEIFQGLDILKNQTVRGG

TVEMLFQNLSLIKKYIDRQKEKCGEERRRTRQFLDYLQEFLGVMSTEWAMEG

335：

Human C-IL-5-E: (SEQ ID NO: 335)

ALVGCGGPKPSTPPGSSGGAPASIPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLCTEEIFQGI

GTLESQTVQGGTVERLFKNLSLIKKYIDGQKKKCGEERRRVNQFLDYLQEFLGVMNTEW IIES

336：

Human C-IL-5-F: (SEQ ID NO: 336)

ADPGCGGGGGLAIPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLCTEEIFQGIGTLESQTVQG

GTVERLFKNLSLIKKYIDGQKKKCGEERRRVNQFLDYLQEFLGVMNTEWIIESLEVLAIEGR

337：

Human C-IL-5-S: (SEQ ID NO: 337)

LACGGGGGIPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNHQLCTEEIFQGIGTLESQTVQGGT

VERLFKNLSLIKKYIDGQKKKCGEERRRVNQFLDYLQEFLGVMNTEW IIES

338：

Primer NheIL 13-F: (SEQ ID NO: 338)

CTAGCTAGCCGGGCCGGTGCCAAGATC

339：

Primer XhoIL 13-R: (SEQ ID NO: 339)

TTTCTCGAGGAAGGGGCCGTGGCGAA

340：

Primer Spelinker 3-F1: (SEQ ID NO: 340)

CCCCGCCGGGTTCTTCTGGCGGTGCTCCGGCTAGCATGGAGATTCCCATGAGCAC

341：

Primer SpeNlinker 3-F2: (SEQ ID NO: 341)

TTTTACTAGTTGGTTGCGGCGGCCCGAAACCGAGCACCCCGCCGGGTTCTTC

342：

Primer IL5 StopXho-R: (SEQ ID NO: 342)

TTTTGCGGCCGCGTTTAAACTCGAGTTATTAGCCTTCCATTGCCCACTC

343：

Primer BamH1-FLK 1-F: (SEQ ID NO: 343)

CGCGGATCCATTCATCGCCTCTGTC

344：

Primer Nhe1-FLK 1-B: (SEQ ID NO: 344)

CTAGCTAGCTTTGTGTGAACTCGGAC

345：

mVEGFR-2(2-3) fragment: (SEQ ID NO: 345)

PFIAS VSDQHGIVYI TENKNKTVVI PCRGSISNLN VSLCARYPEK RFVPDGNRIS WDSEIGFTLP

SYMISYAGMV FCEAKINDET YQSIMYIVVV VGYRIYDVIL SPPHEIELSA GEKLVLNCTA

RTELNVGLDF TWHSPPSKSH HKKIVNRDVK PFPGTVAKMF LSTLTIESVT KSDQGEYTCV

ASSGRMIKRN RTFVRVHTKP

346

Human C-LT_49-306：(SEQ ID NO：346)

LACGGQDQGRRVEKIIGSGAQAQKRLDDSKPSCILPSPSSLSETPDPRLHPQRSNASRNLASTSQGPVAQSSR

EASAWMTILSPAADSTPDPGVQQLPKGEPETDLNPELPAAHLIGAWMSGQGLSWEASQEEAFLRSGAQFSP

THGLALPQDGVYYLYCHVGYRGRTPPAGRSRARSLTLRSALYRAGGAYGRGSPELLLEGAETVTPVVDPIG

YGSLWYTSVGFGGLAQLRSGERVYVNISHPDMVDYRRGKTFFGAVMVG

347

Human C-LT _126-306：(SEQ ID NO：347)

LACGGSPAADSTPDPGVQQLPKGEPETDLNPELPAAHLTGAWMSGQGLSWEASQEEAFLRSGAQFSPTHGL

ALPQDGVYYLYCHVGYRGRTPPAGRSRARSLTLRSALYRAGGAYGRGSPELLLEGAETVTPVVDPIGYGSL

WYTSVGFGGLAQLRSGERVYVNISHPDMVDYRRGKTFFGAVMVG

348

Modified human prion protein fragment: (SEQ ID NO: 348)

VGGLGGYMLGSAMSRPIIHFGSDYEDRYYRENMHRYPNQVYYRPMDE

YSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQ

RGRLAGGGGCG

349

Modified bovine prion protein fragment: (SEQ ID NO: 349)

VGGLGGYNLGSAMSRPLlHFGSDYEDRYYRENMHRYPNQVYYRPVDQ

YSNQNNFVHDCVNITVKEHTVTTTTKGENFTETDIKMMERVVEQMCITQYQRESQAYYQ

RGRLAGGGGCG

350

Modified fragments of sheep prion protein: (SEQ ID NO: 350)

VGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDR

YSNQNNFVHDCVNITVKQHTVTTTTKGENFTETDIKIMERVVEQMCITQYQRESQAYYQ

RGRLAGGGGCG

Claims (9)

1. A composition, comprising:

(a) a non-natural molecular scaffold comprising:

(i) a core particle, and

(ii) an organizer comprising at least one first attachment site, wherein said organizer is attached to said core particle by at least one covalent bond, said core particle being a virus-like particle of a bacteriophage containing a recombinant protein or fragment thereof;

(b) an antigen or antigenic determinant comprising at least one second attachment site,

the second attachment site is selected from:

(i) a non-naturally occurring attachment site for said antigen or antigenic determinant; and

(ii) the naturally occurring attachment site for the antigen or antigenic determinant,

wherein said second attachment site is capable of binding to said first attachment site through at least one non-peptide bond; and

wherein said antigen or antigenic determinant interacts with said scaffold through said binding to form an ordered and repetitive antigen array.

2. A composition, comprising:

(a) a non-natural molecular scaffold comprising:

(i) a core particle selected from the group consisting of:

(1) a core particle of non-natural origin, and

(2) a core particle of natural origin; and

(ii) an formed body comprising at least one first attachment site, wherein the formed body is linked to the core particle by at least one covalent bond;

(b) An antigen or antigenic determinant comprising at least one second attachment site,

wherein said antigen or antigenic determinant is an autoantigen or fragment thereof and said second attachment site is selected from the group consisting of:

(i) a non-naturally occurring attachment site for said antigen or antigenic determinant; and

(ii) the naturally occurring attachment site for the antigen or antigenic determinant,

wherein said second attachment site is capable of binding to said first attachment site through at least one non-peptide bond; and

wherein said antigen or antigenic determinant interacts with said scaffold through said binding to form an ordered and repetitive antigen array.

3. A composition, comprising:

(a) a non-natural molecular scaffold comprising:

(i) a core particle selected from the group consisting of:

(1) a core particle of non-natural origin, and

(2) a core particle of natural origin; and

(ii) an formed body comprising at least one first attachment site, wherein the formed body is linked to the core particle by at least one covalent bond; wherein said core particle is a virus-like particle comprising bacteriophage recombinant proteins or fragments thereof;

(b) an antigen or antigenic determinant comprising at least one second attachment site,

The second attachment site is selected from:

(i) a non-naturally occurring attachment site for said antigen or antigenic determinant; and

(ii) the naturally occurring attachment site for the antigen or antigenic determinant,

wherein said second attachment site is capable of binding to said first attachment site through at least one non-peptide bond; and

wherein said antigen or antigenic determinant interacts with said scaffold through said binding to form an ordered and repetitive antigen array.

4. A composition comprising influenza M2 protein or a fragment thereof that has been linked by a covalent bond to a protein selected from the group consisting of phage coat protein, bacterial pili, HbcAg, and fragments thereof.

5. A pharmaceutical composition comprising

a) The composition of any one of claims 1-4; and

b) an acceptable pharmaceutical carrier.

6. A vaccine composition comprising the composition of any one of claims 1-4.

7. A method of producing a non-naturally occurring, ordered, repetitive antigen array comprising:

(a) providing a non-natural molecular scaffold comprising:

(i) a core particle selected from:

(1) a core particle of non-natural origin; and

(2) a core particle of natural origin; and

(ii) An formed body comprising at least one first attachment site, wherein the formed body is linked to the core particle by at least one covalent bond; and

(b) providing an antigen or antigenic determinant comprising at least one second attachment site, wherein said antigen or antigenic determinant is an autoantigen or fragment thereof, and said second attachment site is selected from the group consisting of:

(i) a non-naturally occurring attachment site for said antigen or antigenic determinant; and

(ii) the naturally occurring attachment site for the antigen or antigenic determinant,

wherein said second attachment site is capable of binding to said first attachment site through at least one non-peptide bond; and

(c) binding said non-native molecular scaffold and said antigen or antigenic determinant,

wherein said antigen or antigenic determinant interacts with said scaffold through said binding to form an ordered and repetitive antigen array.

8. A method of producing a non-naturally occurring, ordered, repetitive antigen array comprising:

(a) providing a non-natural molecular scaffold comprising:

(i) a core particle; and

(ii) an formed body comprising at least one first attachment site, wherein the formed body is linked to the core particle by at least one covalent bond; said core particle is a virus-like particle comprising bacteriophage recombinant proteins or fragments thereof;

(b) Providing an antigen or antigenic determinant comprising at least one second attachment site selected from the group consisting of:

(i) a non-naturally occurring attachment site for said antigen or antigenic determinant; and

(ii) the naturally occurring attachment site for the antigen or antigenic determinant,

wherein said second attachment site is capable of binding to said first attachment site through at least one non-peptide bond; and

(c) binding said non-native molecular scaffold and said antigen or antigenic determinant,

wherein said antigen or antigenic determinant interacts with said scaffold through said binding to form an ordered and repetitive antigen array.

9. A capsid-forming coat protein comprising a mutant Q β coat protein having an amino acid sequence selected from the group consisting of seq id no:

a) amino acid sequence SEQ ID NO: 255;

b) amino acid sequence SEQ ID NO: 256 of;

c) amino acid sequence SEQ ID NO: 257;

d) amino acid sequence SEQ ID NO: 258; and

e) amino acid sequence SEQ ID NO: 259.