JP2011115042A - HBs-PEPTIDE CONJUGATE - Google Patents

Abstract

Disclosed is a peptide particle that can be used as a vaccine, wherein a foreign peptide such as an antigenic determinant of a pathogen causing an infection is expressed on the surface thereof.
HBs protein is a kind of membrane protein, but is self-assembled and has strong membrane-type particle-forming ability. Therefore, in order to construct an HBs peptide fusion in which a foreign peptide (for example, an epitope of a pathogen) is bound to the N-terminus of the HBs protein, DNA encoding them is translated from this peptide to HBs as a single protein. When incorporated into a vector and expressed, HBs particles in which a foreign peptide is expressed on the surface are generated.
[Selection] Figure 2

Description

Hepatitis B virus (Non-patent Document 1, etc.) has a spherical shape, and core particles containing viral nucleic acids are covered with surface proteins unique to the virus. This surface protein consists of three membrane proteins (Large S, middle S, and small S). Among these, the smallest small S (hereinafter referred to as “HBs particles”) is self-assembled and has a strong membrane-type particle ability. (FIG. 1, Non-Patent Document 2).
On the other hand, conventional vaccines are designed to inactivate infectious pathogens for the purpose of forming neutralizing antibodies against individual pathogens, or to infect pathogen gene product epitope peptides or wild type pathogens. It is a live attenuated vaccine that can be passaged in the laboratory, but it must be individually examined according to each pathogen or its antigen, and it takes time and money to produce it. It often requires special facilities to handle type pathogens and, as a result, is often less effective.

Nucleic Acid Research 11: 4601-4610, 1983 Journal of Virology, 1991, 65 (7), 4521-3529

  The present invention provides peptide particles that can be used as a vaccine, on the surface of which foreign peptides such as antigenic determinants of pathogens that cause infections are expressed.

HBs protein is a kind of membrane protein, but self-assembles to form membrane-type particles. Using this property, in order to construct an HBs peptide fusion in which a foreign peptide (for example, an epitope of a pathogen) was bound to the N-terminus of the HBs protein, the DNA encoding this foreign peptide and the downstream thereof were arranged. When the HBs gene was incorporated into a vector so as to be translated as one protein and expressed, formation of HBs particles in which a foreign peptide was expressed on the surface was confirmed.
In addition, if a similar vector is constructed by providing a cloning site for inserting any DNA into this foreign peptide part, HBs particles in which any peptide such as a pathogen epitope is expressed on the surface can be easily formed. Can do.

That is, the present invention comprises an HBs protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in these amino acid sequences, and has particle forming ability It is an HBs peptide fusion that is formed by binding an exogenous peptide to the N-terminus of the HBs protein it has, and has a particle shape.
The present invention also comprises an HBs protein comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in these amino acid sequences and has particle-forming ability. It is an HBs peptide fusion that is formed by binding a foreign peptide to the N-terminus of the HBs protein, has a particle shape, and exhibits immunogenicity by the peptide.
Furthermore, the present invention is a vaccine for the prevention or treatment of infectious diseases, wherein the foreign peptide contains the antigenic determinant of the pathogen that causes the infectious disease, and consists of the above HBs peptide fusion.
Furthermore, the present invention has a promoter, a cloning site for inserting a DNA encoding a foreign peptide arranged downstream thereof, and an HBs gene having the nucleotide sequence of SEQ ID NO: 3 or 4 arranged downstream thereof. An expression vector in which the DNA encoding this peptide and the HBs gene are incorporated so as to be translated as one protein.

  By utilizing the technology of the present invention, a basic expression vector for artificially expressing an epitope of a heterologous pathogen on the outer surface of the HBs particle is constructed, and the HBs particle having a pathogen epitope on the particle is replaced by replacing the epitope part. Since it can be created, it is possible to create a basic and universal vaccine capable of dealing with various pathogen epitopes without requiring a special facility. Since this vaccine is in the form of particles, it is assumed that the vaccine is multivalent and has a high titer, and it becomes possible to develop a vaccine against HCV and HIV that has been difficult to develop. Moreover, it can be easily transferred to a mass production system using yeast, and a vaccine can be produced at a low cost.

The HBs particle of the present invention is an HBs peptide fusion formed by binding a foreign peptide to the N-terminus of the HBs protein.
The HBs particle is a four-transmembrane membrane protein composed of 226 amino acids, and self-assembles to have a particle forming ability (Non-patent Document 3).
Two clones of adr4 (SEQ ID NO: 1, 3, NCBI HBVADR4 X01587) and adw2 (SEQ ID NO: 2, 4, NCBI HBVADW2 X02763) are known as HBs proteins. All of these have the ability to form particles having a particle size of 15 to 25 nm.
When an arbitrary peptide (foreign peptide) is bound to the N-terminus of the HBs protein, as shown in FIG. 2, the fusion protein forms particles having a particle size almost the same as that of the HBs particles (about 15 to 25 nm). This added peptide is expressed on the surface of the particle.
The foreign peptide to be bound to the N-terminus of the HBs protein is preferably 20 to 70 amino acids. If it is larger than this size, it may affect particle formation, and if it is smaller than this size, the activity as an antigen may be reduced.

The foreign protein may contain an antigenic determinant of a pathogen that causes infection. Such pathogens include hepatitis C virus (HCV) (NCBI HPCHCPO, NCBI AB080299), human immunodeficiency virus (HIV) (NCBI AB077816, NCBI NC_001802), malaria or avian influenza antigens (NCBI CY030567, NCBI CY030565, DQ489691 ), Cancer antigens that may be identified in the future. A vaccine production system can be established by building a similar system for these. If you are not sure where the required epitope is, insert it a little longer, including the expected portion.
It is also possible to prepare fusion HBs proteins having different peptides (epitope) and form particles to create a multi-antigen vaccine, which can cope with pathogenic epitopes with severe mutations.

In order to express such fusion protein particles, an appropriate expression vector can be constructed.
This vector may be any commonly used vector such as a plasmid, phage, virus or the like.
Such a vector has a promoter, an HBs gene having a base sequence of SEQ ID NO: 3 or 4 arranged downstream thereof, and a DNA encoding the foreign peptide arranged downstream thereof.
The promoter is not particularly limited as long as it has an activity of initiating transcription into RNA in the vector. As the promoter, for example, cytomegalovirus immediate early gene enhancer / promoter, SV40 enhancer, retrovirus LTR, polyhedron promoter in insect cells and PHO5 promoter in yeast can be preferably used in mammalian cells.

It is necessary to integrate the DNA encoding the peptide to the HBs gene so as to be translated as one protein. That is, it is required that the HBs gene is incorporated from the DNA encoding this peptide so as to fuse in-frame. Specifically, there is no termination codon between these genes, and the genetic code is It needs to be placed on a vector so that it can be read without causing a frame shift.
The size that can be inserted as DNA encoding this foreign peptide is preferably about 60 to 210 bp.
Further, a sequence having a cloning site for inserting this DNA may be used in place of the DNA encoding this foreign peptide.

Examples of regions other than the above-described components contained in the vector of the present invention include, for example, enhancers, transcription regulatory regions, transcription termination sequences, replication origins, drug resistance genes used as selection markers, and regulatory genes necessary for operator functions. Etc. may be included.
This enhancer may be a cytomegalovirus immediate early gene enhancer / promoter in mammalian cells, SV40 enhancer, retrovirus LTR, polyhedron promoter in insect cells, PHO5 promoter in yeast, etc.
The regulatory region is not particularly limited as long as it can control the expression of a gene located downstream of the promoter. For example, a lac operator derived from E. coli lactose operon can be used. Such an operator sequence is usually arranged in the vicinity of the transcription start point downstream of the promoter.

  In addition, as an expression vector used in the present invention, an expression vector using cultured mammalian cells, an expression vector using insect cells, an expression vector using yeast, and the like can be used. In particular, HBs protein can be expressed in yeast, and since it is actually applied clinically as a currently used HBV vaccine, it is extremely easy to introduce this technology into a yeast expression system, and mass production can be established in a short time. it can.

  When an expression vector (plasmid) using mammalian cultured cells is used, a mammalian expression plasmid having a mammalian enhancer-promoter-foreign peptide fusion HBs gene-poly A addition signal as a component is constructed. The enhancer-promoter can be optionally replaced depending on the cell type to be expressed. An actual vaccine production vector may be obtained by inserting a pathogen epitope into a foreign peptide portion to form an HBs fusion type. Such epitopes are synthesized based on nucleotide sequence information using primers (sometimes with restriction enzyme sites added) and cloned plasmids or pathogen genomes collected from infected patients or cDNA from production RNA as templates. Can be synthesized by PCR. Primers may also be synthesized for the HBs translation region and synthesized by PCR using the cloned plasmid as a template. The epitope and HBs are constructed so as to fuse in-frame, and inserted into an HBs fusion gene cassette construction vector (FIG. 3b). To the expression vector, the epitope-HBs part is excised with a restriction enzyme and inserted into the expression vector shown in FIG. 3a.

When an expression vector (plasmid) using insect cells is used, an insect cell expression vector (plasmid) is obtained by using an insect cell enhancer-promoter-epitope fusion HBs gene-insect cell transcription termination sequence.
Moreover, when using the expression vector (plasmid) using yeast, it is set as the yeast expression vector (plasmid) by setting it as the yeast enhancer-promoter-epitope fusion HBs gene-yeast transcription termination sequence.

In order to obtain the HBs peptide fusion of the present invention and particles thereof, for example, the above expression vector is appropriately introduced into a host by transfection or transformation, and stable expression using a transient expression system or drug resistance gene activity is used. A system is established and expression in a stable expression strain is confirmed by an immunofluorescent antibody method or the like. The fused HBs particles secreted into the culture supernatant or into the solution by cell disruption are purified and concentrated by ammonium sulfate precipitation, gel filtration, ultracentrifugation, and affinity column method.

The following examples illustrate the invention but are not intended to limit the invention.

In this example, a fusion protein in which a peptide (HisHBs, 65 amino acids) containing a histidine hexamer (His6) as a foreign peptide was bound to the N-terminus of the HBs protein (SEQ ID NO: 1) was expressed as a particle. It was confirmed that the surface properties were examined.
(1) Cloning of HBs gene
SS5'FW;5'-AAGTCGAC (SalI) ATGGAGAACACAACATCAGGA-3 '(SEQ ID NO: 7) primer using 10 ng of the plasmid pBR322? HBVadr4 (Hamamatsu Medical University Infectious Diseases possessed) cloned from HBV genomic DNA (SEQ ID NO: 3) And SS3′RV; 5′-CACCGCGG (SacI) TTTATTAGGGTTTAAATGTAT-3 ′ (SEQ ID NO: 8) primers, digested with restriction enzymes SalI and SacI and cloned into the same site of pbluescriptII to obtain pBSII-HBs. It was confirmed that the nucleotide sequence was not different from the original clone.
(2) Cloning of peptide containing histidine hexamer (His6)
pEBVHisA vector (Hamamatsu Medical University Infectious Diseases possession) 10ng as a template His5'FW;5'-AACTCGAGT (XhoI) CTCATCATCATCATCATCATCATGGT-3 (SEQ ID NO: 9) 'primer and His3'RV;5'-AACTCGAG (XhoI) GGATCGATCCGGCCTGCCGGCCT Amplified with a −3 ′ (SEQ ID NO: 10) primer, digested with the restriction enzyme XhoI, and then inserted into the XhoI of the pBSII-HBs obtained above to obtain the HBs fusion gene cassette construction vector pBSII-His6HBs. It was confirmed by nucleotide sequence analysis that the nucleotide sequence of the gene was not different from the original sequence and that the reading frame was fused (SEQ ID NOs: 5 and 6, FIG. 4).

(3) Construction of mammalian expression vector After digesting the pBSII-His6HBs obtained above with the restriction enzymes KpnI and SacI, the fragments excised by agarose electrophoresis were separated, and the His6HBs fragment was purified from the gel. The fragment ends were flattened with T4 DNA polymerase and cloned into the PvuII site of pREP4 (Hamamatsu Medical University Infectious Diseases) to obtain pREP4-His6HBs. The directionality of the gene was confirmed by base sequence analysis.
(4) Expression in cultured mammalian cells 5 μg of the pREP4-His6HBs obtained above was transfected into a cultured cell HEK293 (Hamamatsu Medical University Infectious Diseases owned) 2 × 106/10 cm dish. After collecting 10 ml of the transient expression culture supernatant 2 days after transfection, the colonies that appeared after selection with a culture solution containing 0.5 mg / ml hygromycin were isolated, and 6 His6HBs / 293 cell clones were established. Products expressed in the cytoplasm were confirmed by immunofluorescence antibody method using anti-His6 antibody.
(5) Separation and concentration of His6HBs in culture supernatant 20 μl of Ni-NTA agarose was added to 50 ml of the above transient expression culture supernatant and His6HBs / 293 cell culture supernatant and allowed to react at room temperature for 30 minutes. Ni-NTA agarose was precipitated by centrifugation at 3000 xg. A 100 μl portion of the supernatant fraction was collected as an unbound fraction (UB). Wash the Ni-NTA agarose (50 mM NaPO ₄ ^2- , 300 mM NaCL, 20 mM imidazole, pH 8.0) twice with 5 ml, then eluate (50 mM NaPO ₄ ^2- , 300 mM NaCl, 300 mM imidazole, pH 8.0) Elute twice with 100 μl to obtain E1 and E2.

(6) Confirmation of separation and concentration of His6HBs 10 μl each of the unbound fraction (UB) and elution fraction (E1, E2) obtained above were processed for protein electrophoresis SDS-PAGE, followed by Western blotting after SDS-PAGE. Transferred to PVDF membrane for analysis. After the transfer, the membrane was immersed in a buffer containing 10% dry milk (TBS-T; 50 mM Tris-HCl, 150MM NaCl, 0.1% Tween20) for 1 hour for blocking operation. After completion, wash 3 times with TBS-T, react with TBS-T containing primary antibody mouse monoclonal anti-His6 antibody (Nacalai Tesque) (100 ng / ml) for 3 hours, then wash 3 times with TBS-T. The secondary antibody HRP-conjugated sheep anti-mouse IgG antibody (manufactured by GE Healthcare) was reacted with TBS-T containing 10,000 dilutions for 1 hour. After washing with TBS-T three times, the reaction product was immersed in an HRP luminescence reaction solution and detected with a chemiluminescence detector (Fuji Film LAS3000). The result is shown in FIG. Protein molecules that react with the anti-His6 antibody in the eluted fractions (E1, E2) were detected. This result means that His6HBs is expressed and secreted in a form that recognizes at least His6.
(7) Proof that His6HBs forms particles
It was confirmed by sucrose density gradient centrifugation that HisHBs separated and concentrated with Ni-NTA agarose actually formed particles. 300 μl of the fraction separated and concentrated with the above Ni-NTA agarose is layered on top of 700 μl sucrose 60%, 10% from the bottom, and centrifuged for 20 hours at 4 ° C. in an ultracentrifuge Beckman TLS100, TLS55 rotor. 100 μl each was collected. 10 μl of each was processed for protein electrophoresis SDS-PAGE, and transferred to a PVDF membrane after electrophoresis. Thereafter, Western blot analysis was performed using a mouse monoclonal anti-His6 antibody in the same manner as described in (6). As a result, as shown in FIG. 6, protein molecules detected in a fraction corresponding to a density of HBs particles of about 1.20 g / ml were obtained.
The above results indicate that HBs particles having His6HBs on the surface were formed.

It is a conceptual diagram of HBV particle | grains and HBs particle | grains. (A) The particle size of HBV particles is about 45 nm, and (b) the particle size of HBs particles is about 20 nm. It is a figure which shows the mode of particle | grain formation of the fusion protein which couple | bonded the foreign peptide with HBs protein. It is a figure which shows an expression vector and a cassette vector. It is a figure which shows the HBs fusion gene which combined the sequence | arrangement containing histidine hexamer (His6) with the HBs gene. The 7th to 201st positions are a sequence (195 bp) containing histidine hexamer (His6), and the 214th to 894th positions are HBs genes (681 bp). Underline shows restriction enzymes, 1st to 6th and 202th to 207th are XhoI (6bp), 208th to 213th are SalI (6bp), and 895th to 900th are SacI (6bp). The gel-electrophoresis diagram of the precipitate of the Ni-NTA agarose of the culture supernatant of a vector in an Example is shown. E1 and E2 are elution fractions, and UB is an unbound fraction. It is a figure which shows the result of the Western blot using the anti-His6 antibody, and the density measurement of what sucrose density-gradient centrifugation of the HBs peptide fusion in the Example. It can be seen that particles having a density of 1.22 g / ml are formed.

Claims (5)

HBs protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or the N-terminal of HBs protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in these amino acid sequences and having particle-forming ability An HBs peptide fusion comprising a foreign peptide bound to and having a particle shape. HBs protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or the N-terminal of HBs protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in these amino acid sequences and having particle-forming ability A HBs peptide fusion comprising a peptide bound to a foreign peptide, having a particle shape, and immunogenicity due to the peptide. A vaccine for the prevention or treatment of an infectious disease, wherein the foreign peptide comprises an antigenic determinant of a pathogen that causes the infectious disease. It has a promoter, a cloning site for inserting a DNA encoding a foreign peptide arranged downstream thereof, and an HBs gene having the nucleotide sequence of SEQ ID NO: 3 or 4 arranged downstream thereof, and encodes this peptide An expression vector in which DNA and HBs gene are integrated so as to be translated as one protein. The vector according to claim 4, wherein the peptide comprises an antigenic determinant of a pathogen that causes infection.