CN1291233A - Human complement (3-degrading proteinase from i (streptococcus pheumoniae) - Google Patents

Abstract

The present invention relates to the identification and use of a family of human complement C3-degrading proteinases expressed by S. pneumoniae. The proteinase has a molecular weight of about 15 kD to about 25 kD. A preferred proteinase of this invention includes the amino acid sequence of SEQ ID NO:2.

Description

Human complement C3 degrading protease from Streptococcus pneumoniae

field of invention

The present invention relates to Streptococcus pneumoniae and in particular to the identification of a Streptococcus pneumoniae protein capable of degrading human complement protein C3.

Background of the invention

Respiratory tract infections caused by Streptococcus pneumoniae cause an estimated 500,000 cases of pneumonia and 47,000 deaths each year. Those at high risk for bacterial Streptococcus pneumoniae infection are infants under two years of age, individuals with compromised immune systems, and the elderly. In these populations, Streptococcus pneumoniae is the main cause of bacterial pneumonia and meningitis. Furthermore, Streptococcus pneumoniae is the leading cause of ear infections in children of all ages. Both children and the elderly are impaired in the synthesis of protective antibodies to the capsular polysaccharide of S. pneumoniae following bacterial colonization, local or systemic infection or following immunization with purified polysaccharide. Streptococcus pneumoniae is a major cause of invasive bacterial respiratory disease in both HIV-infected adults and children, and causes hemorrhagic infections in these patients (Connor et al., Current Topics in AIDS 1987; 1:185-209 and Janoff et al., Ann. Intern. Med. 1992:117(4):314-324).

Individuals who show the greatest risk for severe infection are unable to develop antibodies to existing capsular polysaccharide vaccines. As a result, four conjugate vaccines are currently in clinical trials. Conjugate vaccines include the capsular polysaccharide of S. pneumoniae combined with a protein carrier or adjuvant in the hope of boosting the antibody response. However, conjugate vaccines in clinical trials present other potential difficulties. For example, the S. pneumoniae serotypes most prevalent in the United States are different from those most common in places such as Israel, Western Europe, South Africa or Scandinavia. Thus, a vaccine that is beneficial in one local area may be disadvantageous in another. The potential need to amend currently available capsular polysaccharide vaccines or to develop protein conjugates as capsular vaccines adapted to geographic serotype differences creates prohibitive financial and technical complexities. Therefore, finding immunogenic surface-exposed proteins that are globally conserved among different virulent serotypes is of paramount importance for the prevention of S. pneumoniae infection and for formulating broadly protective S. pneumoniae vaccines. Furthermore, the worldwide emergence of penicillin- and cephalosporin-resistant S. pneumoniae strains the need for an effective vaccine (Baquero et al., J. Antimicrob. Chemother. 1991; 28S; 31-8).

Several S. pneumoniae proteins have been proposed to elicit immune responses against S. pneumoniae in combination with the S. pneumoniae capsular polysaccharide or as single immunogens. It has been reported that the surface proteins associated with the adhesion of Streptococcus pneumoniae to respiratory epithelial cells include PsaA, PspC/CBPll2 and IgAl protease (Sampson et al., Infect. Immun. 1994; 62:319-324, Sheffield et al., Microb. Pathogen .1992; 13:261-9 and Wani et al. Infect. Immun. 1996; 64:3967-3974). Antibodies against these adhesion proteins can inhibit the adhesion of S. pneumoniae to airway epithelial cells and thus reduce invasive propagation. Other cytoplasmic pneumococcal proteins such as pneumolysin, autolysin, neuraminidase, or thioglucosidase have been proposed as vaccine antigens because antibodies can potentially prevent the expression of these proteins in pneumococcal infection. Toxic effects in patients. However, these proteins are not typically located on the surface of S. pneumoniae, but are secreted or released from the bacteria when cells lyse and die (Lee et al., Vaccine 1994; 12:875-8 and Berry et al., Infect. Immun. 1994;62:1101-1108). Although the use of these cytoplasmic proteins as immunogens moderated the late effects of S. pneumoniae infection, antibodies against these white matter neither promoted S. pneumoniae death nor prevented initial or subsequent S. pneumoniae colonization.

The prototype surface protein for which the S. pneumoniae vaccine is being tested is the S. pneumoniae surface protein A (PspA). PspA is a heterologous protein of approximately 70-140 kDa. The structure of PspA consists of an alpha-helix at the amino terminus, followed by a proline-rich sequence, and terminated at the carboxy terminus by a series of 11 choline-binding repeats. Although much information about its structure is known, PspA is not structurally conserved among different S. pneumoniae serotypes, and its function is completely unknown (Yother et al., J. Bacteriol. 1992; 174:601-9 and Yother, J. Bacteriol. 1994;176:2976-2985). Studies have demonstrated the immunogenicity of PspA in animals (McDaniel et al., Microb. Pathogen. 1994; 17:323-337). Although PspA is immunogenic, its ability to act as a vaccine antigen is complicated by the heterogeneity of the PspA lineage, its existence in four structural groups, and its uncharacterized function.

In patients who are unable to develop protective antibodies against the type-specific polysaccharide capsule, the third complement component, C3, and proteins associated with the alternative complement pathway constitute the host's first line of defense against S. pneumoniae infection. Because complement proteins cannot penetrate the strong cell wall of S. pneumoniae, the deposition of opsonic C3b on the surface of S. pneumoniae becomes the main mediator for the clearance of S. pneumoniae. The interaction between S. pneumoniae and C3 in plasma is known to occur during pneumococcal bacteremia, where covalent binding of C3b (the opsonic active fragment of C3) activates recognition and phagocytosis by phagocytes (Johnston et al. 1969; 129: 1275-1290, Hasin HE, J. Immunol. 1972; 109: 26-31 and Hostetter et al., J. Infect. Dis. 1984; 150: 653-61). C3b is deposited on the capsule and cell wall of Streptococcus pneumoniae. However, this method of controlling S. pneumoniae infection is completely ineffective. A method of stimulating the opsonization of S. pneumoniae can ameliorate the disease process caused by this microorganism. There remains a strong need for methods and treatments to limit S. pneumoniae infection.

Summary of the invention

The present invention relates to the identification and use of a group of human complement C3 degrading proteins (proteases) expressed by Streptococcus pneumoniae. The proteins preferably have a molecular weight of about 15 kD to about 25 kD, as determined, for example, on a 10% SDS polyacrylamide gel. The present invention includes a number of proteins that can be isolated from different C3-degrading strains of S. pneumoniae.

In one aspect, the invention relates to an isolated protein having at least 80% sequence identity to SEQ ID NO:2. In a preferred embodiment, the protein is isolated from Streptococcus pneumoniae, or the protein is a recombinant protein. Preferably, the isolated protein degrades human complement protein C3. The preferred protein of the present invention is an isolated protein having an amino acid sequence comprising SEQ ID NO: 2, more preferably the amino acid sequence is SEQ ID NO: 2. The term "isolated" herein means a naturally occurring or synthetic substance that has been removed from its natural environment. The term "protein" herein includes one or more functional units comprising one or more peptides or polypeptides.

The invention also relates to peptides or polypeptides isolated from the C3-degrading proteases of the invention. Preferably, the present invention provides a peptide or polypeptide of at least 15 consecutive amino acids from an isolated protein having 80% sequence identity to SEQ ID NO: 2, and more preferably at least 15 amino acids in SEQ ID NO: 2 A peptide or polypeptide of consecutive amino acids. In another aspect of the invention, the peptide or polypeptide is capable of degrading human complement protein C3.

Preferred embodiments of the present invention include an isolated protein having about 1st to about 58th amino acid in SEQ ID NO: 2 and about 1st to about 174th nucleotide in SEQ ID NO: 1 or its complementary chain . Preferably, the isolated nucleic acid fragment comprises about 150 to about 174 nucleotides of SEQ ID NO: 1 or its complementary strand.

In another aspect, the present invention relates to an isolated protein that degrades human complement protein C3, wherein the nucleic acid encoding the protein can hybridize to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization.

The present invention also relates to an immune system stimulating composition (preferably a vaccine) comprising an effective amount of an immune system stimulating peptide or polypeptide having at least 15 consecutive amino acids derived from a protein, the protein Has at least 80% sequence identity to SEQ ID NO: 2 and is capable of degrading human complement protein C3.

Preferably, the protein is isolated from Streptococcus pneumoniae. In one embodiment, the immune system stimulating composition or vaccine further comprises at least one other immune system stimulating peptide, polypeptide or protein isolated from Streptococcus pneumoniae.

The present invention further relates to an antibody capable of binding (typically specifically binding) to a protein having at least 80% sequence identity to SEQ ID NO: 2 and capable of degrading human complement protein C3. In one embodiment, the antibody is a monoclonal antibody, and in another embodiment, the antibody is a polyclonal antibody. In another embodiment, the antibody is an antibody fragment. The antibody or antibody fragment can be obtained from mouse, rat, goat, chicken, human, or rabbit.

In another embodiment, the antibody is capable of binding to at least a portion of a protein, wherein the nucleic acid encoding the protein can hybridize to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization.

The present invention also relates to isolated nucleic acid fragments (polynucleotides) capable of hybridizing to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. The highly stringent hybridization conditions referred to herein include, for example, hybridization with 6X SSC, 5X Denhardt, 0.5% SDS, and 100 micrograms/ml of fragmented and denatured salmon sperm DNA at 65°C overnight, And once in 2X SSC, 0.1% SDS at room temperature for about 10 minutes, then once at 65 ° C for about 15 minutes, then at least once in 0.2X SSC, 0.1% SDS at room temperature At least 3-5 minutes. In one embodiment, the nucleic acid fragment is isolated from Streptococcus pneumoniae, and in another embodiment, the nucleic acid fragment encodes at least a portion of a protein. In one embodiment, the protein degrades human complement protein C3. In another embodiment, the nucleic acid fragment encodes a peptide or polypeptide that does not degrade human complement C3.

In another embodiment, the nucleic acid fragment is in a nucleic acid vector, and the vector is an expression vector capable of producing at least a portion of a protein. Cells comprising the nucleic acid fragment are also contemplated in the present invention. In one embodiment, the cell is a bacterial or eukaryotic cell.

The present invention further relates to an isolated nucleic acid fragment comprising the nucleic acid sequence of SEQ ID NO: 1 or its complementary strand. The present invention further relates to RNA fragments transcribed from double-stranded DNA comprising SEQ ID NO:1.

In another aspect of the present invention, the present invention relates to a method of generating an immune response against Streptococcus pneumoniae in a mammal, particularly a human, comprising the step of administering to the animal a composition to generate an immune response against the protein , the composition comprises at least a portion of a protein in a therapeutically effective amount, wherein the nucleic acid encoding the protein can hybridize to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. The immune response can be a B cell response, a T cell response, an epithelial cell response or an endothelial cell response. In a preferred embodiment, the composition is a vaccine composition. It is preferred that the protein is at least 15 amino acids in length, and it is also preferred that the composition further comprises at least one other immune system stimulating peptide, polypeptide or protein from S. pneumoniae. In one embodiment, the protein includes at least 15 amino acids of SEQ ID NO:2.

The present invention further relates to an isolated protein of about 15 kDa to about 25 kDa from Streptococcus pneumoniae capable of degrading human complement C3, and to a method for inhibiting Streptococcus pneumoniae-mediated C3 degradation, comprising the steps of: The cocci bacterium is exposed to an antibody capable of binding to a protein having at least 80% sequence identity to SEQ ID NO:2.

The present invention also relates to a method for inhibiting C3-mediated inflammatory response and xenotransplantation rejection, which comprises expressing a protein having the amino acid sequence of SEQ ID NO: 2 on the surface of an animal organ used for xenotransplantation. This approach is particularly advantageous for eliciting expression of the proteins of the invention in, for example, porcine kidneys, and thereby suppressing C3-mediated inflammation following xenotransplantation.

The present invention also relates to an isolated nucleic acid molecule comprising a region of at least 15 nucleotides capable of hybridizing to at least one region of SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. In one embodiment, the isolated nucleic acid molecule hybridizes to at least a region of SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. Preferably, at least one region comprises nucleotides 1-174 or 320-492 of SEQ ID NO:1.

In another embodiment, the present invention also relates to an isolated nucleic acid molecule comprising a region of at least 15 nucleotides capable of hybridizing to at least one region of SEQ ID NO: 4 or its complementary strand under highly stringent hybridization conditions hybridize. In one embodiment, the isolated nucleic acid molecule hybridizes to at least a region of SEQ ID NO: 4 or its complementary strand under highly stringent hybridization conditions. Preferably, at least one region comprises nucleotides 507-681 or 827-999 of SEQ ID NO:4.

In another embodiment, at least a portion of the nucleic acid molecule of SEQ ID NO: 4 encodes at least a portion of a protein. Preferably, the portion has a predicted amino acid sequence as shown in SEQ ID NO: 5 and has a molecular weight of about 75 kDa to about 85 kDa as determined by, for example, SDS-PAGE.

The present invention also relates to isolated DNA fragments or primers having nucleic acid sequences such as SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

In another aspect of the present invention, the present invention relates to an immune system-stimulating composition or vaccine comprising a therapeutically effective amount of at least a portion of a protein, wherein the nucleic acid encoding the protein is compatible with SEQ ID NO: 4 or its Complementary strands hybridize under high stringency hybridization conditions. The present invention relates to a method for generating an immune response against Streptococcus pneumoniae in mammals, especially humans, comprising the step of administering to the animal a composition comprising a therapeutically effective At least a portion of a protein of which the nucleic acid encoding the protein can hybridize to SEQ ID NO: 4 or its complementary strand under highly stringent hybridization conditions.

In another embodiment, the invention relates to a vaccine or immune system-stimulating composition comprising a therapeutically effective amount of at least a portion of a protein, wherein the protein is effective for immunizing Or treat mammals against infection or invasive reproduction of Streptococcus pneumoniae. The protein is derived from a nucleic acid molecule capable of hybridizing to the nucleic acid sequence shown in SEQ ID NO: 4 or its complementary strand under highly stringent hybridization. Preferably, the protein is provided in an amount effective to provide a therapeutic effect on the mammalian subject, especially a human subject.

Brief description of the diagram

Fig. 1 provides the nucleic acid sequence (SEQ ID NO: 1) of the translation part of a C3-degrading protease gene of the present invention.

Figure 2 provides the amino acid sequence (SEQ ID NO: 2) of a C3-degrading protease gene of the present invention.

Figure 3 provides the amino acid sequence of a C3-degrading protease gene of the present invention, which is juxtaposed with the nucleic acid sequence encoding a C3-degrading protease gene of the present invention (SEQ ID NO: 2-3).

Figure 4 provides the nucleic acid sequence (SEQ ID NO: 4) of the predicted 79kDa amino acid sequence.

Figure 5 provides the predicted 79 kDa amino acid sequence (SEQ ID NO: 5).

Figure 6 shows the sequence alignment of SEQ ID NO: 1 and SEQ ID NO: 4.

Figure 7 shows the sequence alignment of SEQ ID NO: 2 and the corresponding amino acids 169-331 in SEQ ID NO: 5.

Detailed Description of Preferred Examples

The present invention relates to the identification and isolation of human complement C3-degrading proteases and nucleic acids encoding C3-degrading proteases with a molecular weight of approximately 20 kDa (±5 kDa) on a 10% SDS-PAGE gel. It has been observed that log-phase cultures of S. pneumoniae from several serotypes degrade first the α-chain of C3 and then the β-chain, without producing a well-defined C3 cleavage fragment (Angel et al., J. Infect. Dis. 170:600-608, 1994). This non-cleavage degradation pattern is substantially different from the products of other microorganisms, such as the elastase of Pseudomonas aeruginosa and the cysteine protease of Entamoeba histolytica.

The term "degradation" as used herein means the removal of amino acids from proteinaceous molecules to produce peptides or polypeptides. As observed on polyacrylamide gel, the protein of the present invention degrades C3 without generating specific cleavage fragments. The C3-degrading proteases of the invention are somewhat better for C3, eg, the C3-degrading proteases do not degrade other proteins such as albumin.

A C3-degrading protease of approximately 20 kDa was isolated from an insertionally interrupted S. pneumoniae gene library identifying clones with attenuated C3-degrading activity compared to wild-type S. pneumoniae. Example 1 provides an exemplary assay for assessing the C3-degrading activity of clones. Clones with reduced C3 degrading activity were identified and a 546 bp SmaI insert was selected based on the clones showing reduced C3 degrading activity. This SmaI fragment was used as a probe for the S. pneumoniae gene library prepared from the CP1200 strain. Positive clones from the S. pneumoniae gene bank that hybridized to the SmaI fragment were isolated and the open reading frames of genes associated with C3-degrading activity were identified. The following oligonucleotide (SEQ ID NO: 10) having sequence identity to a portion of PspA was used in a differential hybridization reaction to demonstrate that the gene encoding the C3-degrading protease is distinct from the gene encoding PspA.

SEQ ID NO: 10

GAAAACAATAATGTAGAAGACTACTTTTAAAGAAGCTTTAGA

The complete open reading frame of the 20kDa protein has 492 base pairs (SEQ ID NO:1), and the predicted molecular weight is about 20kDa (+/-5kDa) or about 163 amino acids (SEQ ID NO:2). An exemplary gene sequence encoding a C3-degrading protein is shown in Figure 1 (SEQ ID NO: 1), and the amino acid sequence of the protein is shown in Figure 2 (SEQ ID NO: 2). Figure 3 combines the optimal gene sequence and the corresponding optimal translation protein into SEQ ID NO: 3.

Using SEQ ID NO: 2, the amino acid sequence of this protein was determined to be unrelated to other proteins in the GenBank or Swiss Prot databases. The predicted protein has the proline-rich sequence characteristics of prokaryotic cell membrane proteins (especially between amino acids 80-108), which suggests that the protein is expressed on the cell surface. The amino acid sequence does not show significant choline-binding repeats. The Streptococcus pneumoniae lysate and supernatant from the CP1200 culture were electrophoresed on an SDS-PAGE gel infiltrated with C3, and a band of about 20 kDa (± 5 kDa) was identified in both the supernatant and the lysate ( band), confirming that a protein of the size predicted by SEQ ID NO: 2 possesses C3-degrading activity (see Example 2). As described in Example 3, the gene encoding the 20 kDa C3-degrading protease was present in at least 24 S. pneumoniae isolates representing at least five serotypes (serotypes 1, 3, 4, 14, and 19F) Conservative.

The full-length gene encoding the C3-degrading protease of the present invention is inserted into a gene expression vector for expression in Escherichia coli (E. coli). Recombinant C3-degrading proteases were isolated as described in the Examples. Those of ordinary skill in the art will appreciate that for a particular gene sequence (as provided in SEQ ID NO: 1), different expression vectors can be used to express the gene. Furthermore, various well-known methods in the art can be used to produce and isolate the recombinant protein of the present invention, and those of ordinary skill in the art will also understand that the C3 degradability assay of the present invention will measure expression Whether a specific expression system other than the one described above is functional does not require further experimentation. Many different molecular and immunological techniques can be found in the basic technical literature, such as Sambrook et al. (Molecular Cloning-A Laboratory Manual, 1989 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Harlow et al. (Antibodies; A Laboratory Manual ．Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press, 1988).

The gene encoding the C3-degrading protein of the present invention is identified by using the plasmid gene library prepared from the genomic DNA fragment of Streptococcus pneumoniae from CP1200 strain. Although many different methods are known to obtain a plasmid gene bank, in a preferred strategy, the construction of the plasmid gene bank will come from Streptococcus pneumoniae CP1200 strain (obtained from D.A.Morrison, University of Illinois, Champagne- Urbana, Illinois, and a Sau3A-cut S. pneumoniae genomic DNA fragment (0.5-4.0 kb) as described by Havarstein LF et al. ), inserted into the BamHI position of an insertable shuttle (shuttle) vector pVA891 (erm ^r , cm ^r ; with the replication source of Escherichia coli). The gene library was transformed into Escherichia coli DH5αMCR strain by electroporation. Plasmids were extracted from some randomly selected E. coli transformants, and it was shown that all transformants contained recombinant plasmids.

Plasmid gene bank DNA can be extracted from E. coli transformants and used to transform the CP1200 parental S. pneumoniae strain using insertional mutagenesis by isosteric recombination.

S. pneumoniae strain CP1200 cells can be converted into competent cells in CTM medium using a pH shift with HCl procedure. Competent cells were stored in aliquots at -70°C until needed.

The isolated protein of the present invention was incubated with human complement C3 in the presence of PBS at 37° C. for 4 hours to detect C3 degradation. A control sample without isolated S. pneumoniae protein can serve as a control group for comparative purposes.

The protein of the present invention has an apparent molecular weight of about 20 kDa (±5 kDa) on a 10% SDS-polyacrylamide gel and preferably has a molecular weight of about 15 kDa to about 25 kDa. An exemplary protein sequence is shown in SEQ ID NO:2. Those of ordinary skill in the art will appreciate that some variations in amino acid sequence are to be expected and such variations should not be excluded from the scope of the present invention. For example, conservative mutations are not excluded from the scope of the invention, nor are variations in amino acid sequence identity of less than about 80% amino acid sequence identity (and preferably less than about 90% amino acid sequence identity). Excluded within the scope of the invention are those where the protein is capable of degrading human complement protein C3 and in particular where the protein is isolated or originally obtained from a Streptococcus pneumoniae bacterium. Fragments of this protein are also within the scope of the invention, particularly when these fragments are capable of degrading human complement protein C3.

Some nucleic acid sequence variation can be expected among S. pneumoniae strains and serotypes, as can some amino acid variation. Conservative amino acid substitutions are known in the art and include, for example, substitutions with other members of the same class as the amino acid. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, and tryptophan. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such changes are not expected to affect molecular weight or isoelectric point as determined by polyacrylamide gel electrophoresis. Particularly preferred conservative substitutions include, but are not limited to, substitution of Lys for Arg (and vice versa) to maintain a positive charge; substitution of Glu for Asp (and vice versa) to maintain a negative charge; substitution of Ser for Thr is to maintain the free state OH; and Gln is replaced by Asn to maintain the free state NH ₂ .

Preferred proteins of the present invention include those having the amino acid sequence of SEQ ID NO:2. Other proteins planned in the present invention include proteins that can degrade human complement protein C3, and the nucleic acid encoding the protein can hybridize with SEQ ID NO: 1 under highly stringent hybridization conditions, such as 6X SSC , 5X Denhardt, 0.5% SDS (sodium dodecylsulfonate), and 100 μg/ml fragmented and denatured salmon sperm DNA were hybridized overnight at 65 ° C, and once in 2X SSC, 0.1 % SDS at room temperature for about 10 minutes, followed by one wash at 65°C for about 15 minutes, followed by at least one wash in 0.2X SSC, 0.1% SDS at room temperature for at least 3-5 minutes. Typically, the SSC solution contains sodium chloride, sodium citrate and water to prepare a stock solution. Peptides or polypeptides of the protein may also be used. Preferred proteins of the invention include about 1st to about 50th amino acids of SEQ ID NO:2.

The protein of the present invention can be isolated or prepared in protein form. In other words, the nucleic acid encoding the protein or a portion of the protein can be inserted into an expression vector or into the chromosome of a cell so that the protein is expressed in the cell. The protein may be purified from bacteria or another cell, preferably a eukaryotic cell, and more preferably an animal cell. Alternatively, the protein can be isolated from cells expressing the protein, such as S. pneumoniae cells. Peptides or polypeptides are also contemplated in the present invention. The peptide or polypeptide is preferably at least 15 amino acids in length and preferably the peptide or polypeptide has at least 15 consecutive amino acids from SEQ ID NO:2.

Nucleic acids encoding the 20 kDa protein are also part of the present invention. SEQ ID NO: 1 is a preferred nucleic acid fragment encoding a C3-degrading protease. Those of ordinary skill in the art will appreciate that some substitutions do not alter the sequence of the C3-degrading protease to such an extent that the characteristics and properties of the C3-degrading protease are substantially altered. For example, the invention also contemplates nucleic acids having at least 80% identity to SEQ ID NO: 1. The method for determining whether a particular nucleic acid sequence falls within the scope of the present invention should consider whether the particular nucleic acid sequence encodes a C3-degrading protease and has at least 80% nucleic acid identity compared to SEQ ID NO:1. Other nucleic acid sequences encoding C3 proteases include those nucleic acids encoding C3 proteases, wherein the C3 protease has the same sequence or at least 90% sequence identity compared to SEQ ID NO: 2, and includes instances of degeneracy involving the nucleic acid sequences. Degenerate codons mean that different three-letter codons can be used to designate the same amino acid. For example, it is well known in the art that the following RNA codons (and thus, the related DNA codons, wherein T is substituted for U) can be interchanged for encoding each specific amino acid:

Phenylalanine (Phe or F) UUU, UUC, UUA or UUG

Leucine (Leu or L) CUU, CUC, CUA or CUG

Isoleucine (Ile or I) AUU, AUC or AUA

Methionine (Met or M) AUG

Valine (Val or V) GUU, GUC, GUA, GUG

Serine (Ser or S) AGU or AGC

Proline (Pro or P) CCU, CCC, CCA, CCG

Threonine (Thr or T) ACU, ACC, ACA, ACG

Alanine (Ala or A) GCU, GCG, GCA, GCC

Tryptophan (Trp) UGG

Tyrosine (Tyr or Y) UAU or UAC

Histidine (His or H) CAU or CAC

Glutamine (GIn or Q) CAA or CAG

Asparagine (Asn or N) AAU or AAC

Lysine (Lys or K) AAA or AAG

Aspartic acid (Asp or D) GAU or GAC

Glutamic acid (Glu or E) GAA or GAG

Cysteine (Cys or Q) UGU or UGC

Arginine (Arg or R) AGA or AGG

Glycine (Gly or G) GGU or GGC or GGA or GGG

Stop codon UAA, UAG or UGA

Furthermore, a particular DNA sequence can be modified to use codons preferred by a particular cell type. For example, preferred codon usage for E. coli, as well as preferred codon usage for animals, including humans, are known. These alterations are well known to those of ordinary skill in the art, and therefore these gene sequences are considered part of the present invention. Other nucleic acid sequences include nucleic acid fragments of at least 15 (and preferably at least 30) nucleic acids in length from SEQ ID NO: 1, or other nucleic acids of at least 15 (and preferably at least 30) nucleic acids in length Fragment, wherein the fragment can hybridize with SEQ ID NO: 1 under highly stringent hybridization conditions, such as 6X SSC, 5X Denhardt, 0.5% SDS (sodium dodecylsulfonate), and 100 μg/ml of fragmented and denatured salmon sperm DNA was hybridized overnight at 65°C, washed once in 2X SSC, 0.1% SDS at room temperature for about 10 minutes, and then washed once at 65°C for about 15 minutes, This is followed by at least one wash in 0.2X SSC, 0.1% SDS for at least 3-5 minutes at room temperature.

The nucleic acid fragment of the present invention may encode all of SEQ ID NO: 2 or SEQ ID NO: 5, and may not contain SEQ ID NO: 2 or SEQ ID NO: 5 (that is, fragments that cannot be transcribed, including fragments of gene regulatory regions or other similar) or can encode part of SEQ ID NO: 2 or SEQ ID NO: 5, preferably comprising a contiguous nucleic acid fragment encoding at least 9 amino acids from SEQ ID NO: 2 or SEQ ID NO: 5. Since nucleic acid fragments encoding a portion of a C3 protease are contemplated in the present invention, it will be appreciated that not all nucleic acid fragments encode proteins or peptides or polypeptides with C3-degrading activity. Furthermore, nucleic acids of the invention can be mutated to remove (or otherwise inactivate) the C3 degrading activity of the protein. Therefore, the present invention also plans fragments that meet the above hybridization conditions and have C3-degrading activity. Methods of mutating or otherwise altering nucleic acid sequences are well described in the art, and protein production inactivated in terms of immunogenicity and enzymatic properties can be tested for therapeutic use.

The nucleic acid fragments of the present invention can be incorporated into nucleic acid vectors or stably incorporated into the host genome to produce recombinant proteins, including recombinant chimeric proteins. In one embodiment, the C3-degrading protein is encoded by a gene in a vector, and the vector is in the cell. Preferably the cells are prokaryotic cells, such as bacteria. Genes and gene fragments can exist in the form of all or part of the gene fused with another gene, and the C3-degrading protein can exist as a fusion protein of one or more proteins, and the fusion protein is expressed as a single protein. Many different nucleic acid vectors of the invention are known in the art and include many commercially available expression plasmids or viral vectors. The use of such vectors is known to those of ordinary skill in the art. Some exemplary vectors are used in the examples, but should not be considered as limiting the scope of the present invention.

The invention also relates to antibodies that bind (typically specifically) to a protein of about 20 kDa (preferably a protein of about 15 kDa to about 25 kDa) from Streptococcus pneumoniae, and preferably wherein the protein is capable of degrading Human complement C3. Polyclonal antibodies can be raised against all or a portion of the protein. Similarly, monoclonal antibodies can be prepared against all or one of the peptides or polypeptides (fragments) of the about 20 kDa C3 degrading protein of the present invention. Methods for preparing antibodies against proteins are detailed in, eg, Harlow et al., (Antibodies; A Laboratory Manual. Cold Spring Harbor, NY; Cold Spring Harbor Laboratory Press, 1988). In a preferred embodiment, the antibody can be derived from human, rat, mouse, goat, chicken, or rabbit. Protein binding antibody fragments and chimeric fragments are also well known and within the scope of the present invention.

The invention also relates to the use of immunostimulatory compositions. The term "immunostimulatory" or "immune system stimulating" composition means a protein, peptide or polypeptide composition of the invention which activates at least one cell type in the immune system of an individual (e.g. a mammal), preferably , the immunostimulatory composition provides an immunization response or prophylactic effect in normal (uninfected) individuals, typically a vaccine. However, any measurable immune response is beneficial to the individual during a therapeutic application or procedure. Preferred activated cells of the immune system include phagocytic cells, such as neutrophils or macrophages, T cells, B cells, epithelial cells and endothelial cells. Immunostimulatory compositions comprising the peptides, polypeptides or proteins of the invention can be used to raise antibodies in animals such as rats, mice, goats, chickens, rabbits or humans, or to study animals infected by Streptococcus pneumoniae model. Preferred immunostimulatory compositions include an immunostimulatory amount (eg, a therapeutically effective amount) of at least one peptide or polypeptide comprising at least 15 amino acids from the C3-degrading protease.

The term "vaccine" means a composition for immunization. The course of immunization may involve the administration of a protein, peptide, polypeptide, antigen, nucleic acid sequence or complementary (e.g. antisense) sequence, or antibody or suspension thereof, wherein administration of the molecule will result in active immunity and protection against pneumonia Streptococcal infection or invasive reproduction. Typically, such vaccines are formulated as injectable liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The vaccine preparation may optionally be emulsified, or encapsulated in liposomes.

The immunostimulatory composition (such as a vaccine) may further include other proteins in a pharmaceutically acceptable buffer or carrier such as PBS (phosphate buffered saline), or other proteins known in the art. A buffer that is considered suitable and safe for introducing proteins into the host to stimulate the immune system. The immunostimulatory composition may also include other immune system stimulating proteins such as adjuvants or immunostimulatory proteins, peptides or polypeptides from S. pneumoniae or other organisms. For example, a cocktail of peptides or polypeptides may be most beneficial for controlling S. pneumoniae infection. It is preferred to use one or more fragments of the protein of the present invention in a vaccine preparation to resist or limit the invasive propagation of Streptococcus pneumoniae or the pathogenic consequences brought about by the invasive propagation of Streptococcus pneumoniae.

As used herein, "therapeutically effective amount" means an amount effective to produce a desired result. The amount depends on the health or physical (ie, antibody synthesis) condition of the individual's immune system, the degree of protection intended, the dosage form prepared and other relevant factors. It is expected that the serving size will fall within a fairly wide range and can be determined by routine experimentation.

The active immunostimulatory ingredient is often admixed with excipients or diluents which are pharmaceutically acceptable carriers and compatible with the active ingredient. The term "pharmaceutically acceptable carrier" means a carrier that is "acceptable" in the sense of being compatible with the other ingredients of a composition and not hazardous to the recipient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and mixtures thereof. In addition, the immunostimulatory composition (including vaccines) may optionally contain small amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffers, and/or adjuvants that can enhance the effect of the immunostimulatory composition.

Effective adjuvants or carriers include, but are not limited to, aluminum hydroxide, N-acetyl-muramoyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-neo-muramoyl -L-alanyl-D-isoglutamine (CGP11637, known as nor-UDP), N-acetylmurayl-L-alanyl-D-isoglutamyl-L-alanine-2- (1',2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, known as MTP-PE), and RIBI, in 2% squalene/ Tween 80 emulsion contains three components extracted from bacteria, monophosphoryl lipid A, dipycolic acid trehalose and cell wall structure (MPL+TDM+CWS).

The invention also relates to a method of inhibiting Streptococcus pneumoniae-mediated C3 degradation comprising contacting Streptococcus pneumoniae with a protein, such as an antibody, or another protein that binds to an isolated protein from Streptococcus pneumoniae of about 15 kDa to about 25 kDa of protein. The protein capable of binding an isolated protein of about 15 kDa to about 25 kDa can be an antibody or fragment thereof, or the protein can be a chimeric protein comprising an antibody binding region (such as a variable region) from an antibody, the antibody It can specifically recognize the isolated protein of about 15kDa to about 25kDa from Streptococcus pneumoniae and has C3 degradation activity.

The isolated S. pneumoniae protein of the invention can be isolated, optionally purified, and the isolated protein or immunogenic fragment thereof can be used to generate an immunological response, which in one embodiment comprises human or experimental Antibody responses in animals. Peptides or polypeptide fragments of the protein that do not have C3 degrading activity can be tested for their ability to limit the effects of S. pneumoniae infection. Similarly, modification of a protein of the invention may, for example, be via mutation to interrupt or inactivate the C3 degrading activity of the protein. Antibodies capable of inhibiting the C3-degrading activity of the proteins of the invention can be used as a strategy to prevent C3 degradation and facilitate clearance of S. pneumoniae via opsonic pathways. The isolated protein can be used in assays to detect antibodies against Streptococcus pneumoniae, or as part of a vaccine for the treatment of Streptococcus pneumoniae, or as a vaccine comprising one or more proteins, peptides or polypeptides.

Therefore, the term "treating" as used herein refers to a normal mammalian individual or infected with various Streptococcus pneumoniae infections, diagnosed with various S. Prophylaxis or treatment of mammalian subjects. The term "treating" means providing a therapeutic effect to a mammalian individual such that the individual expresses little or no symptoms of S. pneumoniae infection or other related disease. Such treatment may be accompanied by the administration of nucleic acid molecules (sense or antisense), proteins, peptides or polypeptides or antibodies of the invention.

It is further contemplated that the proteins of the invention may be expressed on the surface of vertebrate cells and used to degrade C3, for example, when complement deposition (or activation) is problematic, such as in the case of xenografts or complement-mediated glomerulonephritis . For example, intact S. pneumoniae proteins, recombinant proteins, or portions of either, can be incorporated into xenograft cells and expressed as surface or secreted proteins to prevent complement deposition (and/or complement-mediated inflammatory response) or minimize it.

Another particular aspect of the invention relates to the use of vaccine vectors expressing isolated proteins and peptides or polypeptides derived therefrom. Accordingly, in a further aspect, the present invention provides a method of eliciting an immune response in a mammal comprising providing to a mammal a vaccine vector expressing at least one of the isolated proteins and/or peptides or polypeptides of the present invention or a mixture thereof. animal. The proteins and peptides or polypeptides of the present invention can be delivered to mammals using live vaccine vectors, especially live recombinant bacteria, viruses or other living substances, including when expressing the protein and/or peptide or polypeptide in the form of foreign polypeptides The required genetic material. Especially those bacteria that reproduce in the gastrointestinal tract have been developed as vaccine vectors, such as Salmonella, Shigella, Yersinia, Vibrio, Escherichia Bacteria (Escherichia) and BCG, the above and other examples in J. Holmgren et al., Immunobiol. 184, 157-179 (1992) and J. Discussed in McGhee et al., Vaccine, 10, 75-88 (1992).

Additional embodiments of the invention relate to methods of eliciting an immune response in an individual, such as a mammal, comprising administering an amount of a DNA molecule encoding an isolated protein of the invention and/or a peptide or polypeptide derived therefrom (selectively combined with An infection-facilitating factor) is administered to the individual, wherein the protein and/or peptide or polypeptide retains immunogenicity, and when the protein and/or peptide or polypeptide is incorporated into an immunostimulatory composition (such as a vaccine) and When administered to a human, it provides protection without exacerbation of subsequent infection of the human by the S. pneumoniae pathogen. Infection-facilitating factors are known in the art.

A further plan is that the antisense sequence of the gene encoding the 20kDa protein can be used as a vaccine or therapeutic treatment against Streptococcus pneumoniae infection. Antisense DNA is defined as a non-coding sequence that is complementary to all or part of SEQ ID NO: 1 (i.e. a complementary strand). For example, the antisense sequence of 5'-ATGTCAAGC-3' is 3'-TACAGTTCG-5'. Delivery of antisense sequences or oligonucleotides to animals can cause the animals to produce antibodies, or cause the sequences to be incorporated into live bacteria or other cells such that transcription and/or translation of all or part of the 20 kDa gene product is inhibited.

The antisense nucleic acid sequence can be introduced into Streptococcus pneumoniae or infected cells, for example, by loading the antisense nucleic acid on a suitable carrier (such as liposome). Typically, antisense nucleic acid sequences of 8 or more nucleotides are capable of binding to bacterial nucleic acid or bacterial messenger RNA. Antisense nucleic acid sequences typically comprise at least about 15 nucleotides (preferably at least about 30 nucleotides or more) to provide the desired stability to hybridization products of bacterial nucleic acid or bacterial messenger RNA . The introduction of the sequence preferably inhibits the transcription or translation of at least one endogenous S. pneumoniae nucleic acid sequence. The method of loading antisense nucleic acid is well known in the art, for example U. S． Patent No. 4,242,046.

The present invention also provides a nucleic acid with an open reading frame (SEQ ID NO: 4) of 2163 bases, which includes a nucleic acid with an open reading frame (SEQ ID NO: 1), which encodes a nucleic acid having a molecular weight of about 20 kDa (SEQ ID NO: 2). protein. The 20 kDa protein described herein was then characterized as a C3-degrading protein. Larger open reading frames, eg 2163bp (SEQ ID NO: 4), encode a hypothetical protein of approximately 79 kDa (SEQ ID NO: 5).

All references and publications cited herein are incorporated into this specification as reference materials. There are many different alternative techniques and procedures available to those skilled in the art that would similarly enable a practitioner to successfully implement the contemplated invention based on this specification. Those skilled in the art will appreciate that although the present invention has been described above in terms of specific examples and examples, the present invention is not limited thereto, and many other examples, examples, uses, modifications, and examples within these are possible. Cases other than specific examples, examples, and uses do not depart from the scope of the invention of this application.

Example 1

Identification of intercalation mutants with reduced C3-degrading activity

Insertion mutant lines were obtained from Dr. Elaine Tuomanen (Rockefeller Inst., New York, New York). Clones with inserts were tested in assays for attenuated C3-degrading activity. 137 clones were tested by growing the cells overnight in Todd Hewitt medium on microtiter plates at room temperature. The cells were diluted 1:10 in synthetic medium for S. pneumoniae (see Sicard A.M., Genetics 50:31-44, 1984), and the remaining cells were frozen in microtiter plates. About 200 microliters of diluted in the cell. Cells were incubated at 37°C for 4 hours. 100 microliters of the mixture was added to the ELISA plate and incubated overnight at 4°C. The plates were washed three times with wash buffer and 0.05% Tween 20 in PBS was added to the wells with a 5 min incubation before each wash. 100 microliters of anti-C3 antibody (multi-strain kabe catalase-conjugated goat antibody specific for human C3-IgG fraction, ICN Cappel, Costa Mesa, CA) in 3 liters in PBS %BSA was diluted 1:1200. The ELISA plates were incubated at 37°C in the dark for about 30 minutes to 1 hour and washed with the wash buffer described above. The assay was developed using 12 mg of OPD (in 30 ml of 0.1 M sodium citrate buffer) and 12 microliters of 30% hydrogen peroxide. Assay results were determined by optical density readings at 490 micrometers on an ELISA plate reader.

Each clone was tested four times. Nineteen clones with less than 40% C3 degradation compared to the non-mutated control were selected. These 19 clones were screened six times by the above test, and 6 clones with less than 40% C3 degradation compared with the non-mutated control group were selected from the results. Each of these 6 clones was screened 11 times, and 2 clones with the lowest C3-degrading activity were selected for further study.

A partial sequence of one of the clones was obtained, and a 546 bp SmaI fragment was labeled with 32P random primers (set from Stratagene, LaJolla, CA). The 546 bp SmaI fragment from SEQ ID NO: 1 hybridized on Southern blots to EcoRI and Kpnl treated cuts from a number of S. pneumoniae strains. This same fragment was also used to screen a library of Sau3A fragments obtained from genomic DNA of S. pneumoniae strain CP1200.

A 3.5 kb insert was identified from the CP1200 gene bank. The insert was sequenced and an open reading frame of 492 base pairs including the stop codon was identified. This open reading frame encodes a protein of 163 amino acids with a predicted molecular weight of approximately 18,500 Daltons.

PCR primers were constructed to amplify the open reading frame; the 5' PCR primer included a BamHI position; the 3' primer included a PstI position. The amplified insert was ligated in-frame into the His-tagged E. coli expression vector pQE30 (Qiagen, San Diego, CA). The resulting plasmid was used to transform E. coli strain BL21 (Novagen, Madison, WI) containing the lac repressor plasmid pREP4 (Qiagen). E. coli cultures were induced to express His-tagged proteins and the proteins were column purified with Ni-NTA resin (Qiagen). The purified protein was confirmed by SDS-PAGE gel.

Example 2

Identification of a 20kDa C3-degrading protease

In order to measure the C3-degradability of 20kDa protein, 0.5 mg/ml of C3 (prepared according to Tack et al., Meth. Enzymol. 80:64-101, 1984) was dissolved in dodecyl containing 15% acrylamide. Copolymerization was performed on a sodium sulfonate (SDS) gel (15% SDS-PAGE gel). The supernatant of Streptococcus pneumoniae was obtained from the culture of Streptococcus pneumoniae strain CP1200 grown to the logarithmic phase in Todd Hewitt's medium, and the lysate of Streptococcus pneumoniae was incubated at room temperature for 30 obtained in minutes. The lysate was concentrated 10-fold using a Centricon filter unit (Amicon, Beverly, MA) with a separation value of 10,000 MW. Samples were not heated prior to electrophoresis. Samples of supernatant and lysate were added to a 15% SDS-PAGE gel containing C3, and electrophoresis was performed at 150V at 4°C until the dye front ran off. The gel was washed successively with 50 ml of 2.5% Triton X-100 in water (2 times, 10 minutes), and with 2.5% Triton X-100 in 50Mm Tris-HCl, pH 7.4 (2 times, 10 minutes) and washed with 50mm Tris-HCl, pH 7.4 (2 times, 10 minutes) to remove SDS. After washing, pour 50 ml of 50 mM Tris-HCl, pH 7.4, into the dishes containing these gels, cover the dishes and incubate at 37°C for 1.5 hours and overnight (approximately 16 hours) . The gel was stained with Coomassie blue for 10 minutes and then completely destained.

Against the dark blue background in the lysate and supernatant, two soluble bands can be observed, one of which is approximately 20 kDa in size. C3 protease activity in S. pneumoniae lysates was observed after 1.5 hours of incubation at 37°C, whereas C3 protease activity in Pn supernatants was not observed after overnight incubation. Thus, C3 protease activity is apparently primarily cell-associated.

Example 3

The gene encoding this 20kD protein is conserved in some strains of Streptococcus pneumoniae

Clinical isolates from various strains of Streptococcus pneumoniae (Type 1, Type 3, L002 and L003 (Type 3), Type 4, Type 14 and CP1200, WU2, R6X, 6303, 109, 110 , JY1119, JY182, and laboratory isolates of JY53) to obtain DNA, and use SEQ ID NO: 3 as a probe to detect whether the nucleic acid encoding the 20kD protein is present in the DNA from these strains. Isolated chromosomal DNA was cut with EcoRI and separated via electrophoresis. The DNA was transferred to a solid support and hybridized with the end-marked SEQ ID NO: 3. The hybridization and washing conditions were 6X SSC, 5X Denhardt's, 0.5% SDS and 100 μg/ml of fragmented and denatured DNA. Salmon sperm DNA was hybridized overnight at 65°C, and washed once in 2X SSC, 0.1% SDS at room temperature for about 10 minutes, then once at 65°C for about 15 minutes, then twice in 0.2X SSC, Wash in 0.1% SDS for 3 minutes at room temperature.

The results indicated that SEQ ID NO: 3 also had a hybridization reaction in each tested DNA sample, indicating that the protein was obviously conserved among various bacterial strains. In some strains, the DNA encoding a 20 kDa C3-degrading protein appeared to be part of a larger 2163 bp open reading frame that putatively encodes a 79 kDa protein.

Example 4

Southern blot analysis of Streptococcus pneumoniae DNA/5F1 probe

Five microgram samples of genomic DNA were obtained from 11 S. pneumoniae strains. Each sample was cut with the restriction enzyme KpnI. These samples were then loaded on an agarose gel and resolved by electrophoresis. The samples contained in the gel were then transferred via capillary transfer onto a Hybond-N+ membrane from Amersham (Upsafla, Sweden). A 540 bp SmaI fragment from one of the 5F1 isolates was randomly primed at ³² P using the T7QuickPrime kit (Pharmacia, Piscathaway, NJ) and extracted from non-incorporated nuclei using a NucTrap column (Stragene, La Jolla, CA). Purified from nucleotides and subjected to hybridization.

The hybridization conditions were 6X SSC, 5X Denhardt, 0.5% SDS, and 100 μg/ml fragmented and denatured salmon sperm DNA at 65°C overnight, and once in 2X SSC, 0.1% SDS at room temperature Wash at 0.1% SDS for at least 3-5 minutes at room temperature. The blot showed that the 20kDa gene was present in all tested strains of Streptococcus pneumoniae.

Example 5

Two DNA primers were made from SEQ ID NO: 1 and used to amplify the 20 kDa gene sequence from S. pneumoniae (serotype 3) genobody DNA. The first primer is the 5'-primer (SEQ ID NO: 6), which starts from the ATG start codon of the 20kDa gene, inserts an NcoI position, and has an Ala residue after the ATG start codon to maintain correct reading frame. The second primer was the 3'-primer (SEQ ID NO: 7), which was developed from the stop codon of the 20 kDa gene, where a BamHI position was inserted.

5'-GGGGG CCA TGG CC TCA AGC CTT TTA CGT GAA TTG-3'; (SEQ ID NO: 6)

5’-GGGGG GGA TCC CTA GCT ATA TGA GAT AAA CTT TCC TGC T-3’;

(SEQ ID NO: 7)

The two primers were synthesized in an Applied Biosystems 380A DNA synthesizer (Foster City, CA) using reagents purchased from Glen Research (Sterling, VA). Amplification reactions were performed using a Perkin Elmer Thermocycler (ABI) according to the manufacturer's instructions. The identified PCR products were ligated into the TA-tailed PCR cloning vector pCR2.1 (obtained from Invitrogen, Carlsbad, CA) and used to transform OneShot Top10F'competent cells (Invitrogen). Kanamycin-resistant transformants were screened by restriction enzyme analysis of plasmid DNA prepared by alkaline lysis. An insert fragment of approximately 500 bp was identified and subsequently cut with restriction enzymes NcoI and BamHI. This 500 bp fragment was isolated from a low melting point agarose gel and then ligated into the NcoI-BamHI site of the T7 activating expression vector pET28a from Novagen (Madison, WI).

The ligation mixture was then transformed into Top1OF' cells (Invitrogen), and kanamycin-resistant transformants were selected by restriction enzyme analysis of plasmid DNA prepared by alkaline lysis. The recombinant plasmid (pLP505) was then transformed into BL21 (Novagen) cells and grown in SOB medium supplemented with 30 μg/ml kanamycin. Cells grow to 0. D． A 600 value of 0.6 was followed by induction with 0.4 mMIPTG (Boehringer Mannheirn, Indianapolis, Indiana) for 2-4 hours. Whole cell lysates were prepared and run on a 14% SDS-PAGE gel. Gels were stained with Coomassie blue and expression products were detected. The Coomassie blue-stained gel showed a band between the 28 kDa and 18 kDa molecular weight markers and was determined to be approximately 20 kDa.

The DNA sequence of the insert in the recombinant pLP505 plasmid was obtained using an ABI 370A DNA sequencer. The DNA sequence was compared with the DNA sequence of SEQ ID NO: 1 using Pustell's MacVector DNA matrix dot plot feature (Oxford Molecular Group, Campbell, CA). By comparing the DNA sequences obtained from the pLP505 plasmid, SEQ ID NO: 1 and the genome of Streptococcus pneumoniae (serotype 4), it was shown that the open reading frame (ORF) encoding the 20kD protein may be part of a larger ORF, That is, 2163 bp (SEQ ID NO: 4) encoding a protein with a predicted molecular weight of about 79 kDa in the genome of serotype 4. DNA SEQ ID NO: 4 encodes the predicted amino acid sequence shown in SEQ ID NO: 5.

Streptococcus pneumoniae (serotype 4) genome sequence was obtained from www. tigr. org's Institute for Genomic Research and/or via www. ncbi. nlm. nih. Obtained from NCBI of gov. A sequence comparison was performed between the 20 kDa amino acid sequence (SEQ ID NO: 2) and the predicted 79 kDa amino acid sequence (SEQ ID NO: 5). It can be observed that amino acids 1-58 and amino acids 90-132 in SEQ ID NO: 2 are substantially identical to amino acids 170-227 and amino acids 258-300 in SEQ ID NO: 5, respectively. Proteins and peptides or polypeptides comprising these specific regions are preferred embodiments of the present invention.

Based on the available genomic DNA (serotype 4) sequence, primers flanking the 2163bp ORF were designed and subsequently synthesized using an ABI 380A DNA synthesizer (SEQ ID NO: 8 and 9). SEQ ID NO: 8 is a Streptococcus pneumoniae 5'-primer with an inserted NcoI position and a Glu residue added after the ATG start codon to maintain a correct open reading frame. SEQ ID NO: 9 is a Streptococcus pneumoniae 3'-primer with an inserted HindIII position.

5'-AGA GCT CCT CCC ATG GAA GAT CCG AAT TAT CAG-3'; (SEQ ID NO: 8)

5'-CCG GGC AAG CTT TTA CTT ACT CTC CT-3'; (SEQ ID NO: 9)

A DNA fragment of approximately 2100 bp was subsequently amplified from 4 different S. pneumoniae serotypes (serotypes 3, 5, 6B and 7) to obtain 4 fragments. Each of the four fragments was then ligated into the PCR cloning vector PCR2.1 (Invitrogen) and used to transform OneShot Top10F' cells (Invitrogen). Kanamycin-resistant transformants were screened by restriction enzyme analysis of plasmid DNA prepared by alkaline lysis. A recombinant plasmid containing the PCR product of serotype 7 was identified, for example pLP512. The DNA sequence of the serotype 7 clone was obtained using an ABI model 370A DNA sequencer. The DNA sequence is substantially identical to SEQ ID NO:4 and encodes a predicted amino acid sequence substantially identical to SEQ ID NO:5.

Those skilled in the art will understand that although the present invention has been described above in terms of specific embodiments and embodiments, the present invention is not limited thereto, and many other embodiments, examples, uses, modifications, and implementations are possible. Situations other than these specific examples, examples and uses, without departing from the scope of this application

scope of invention.

Claims (61)

1. An isolated protein, characterized in that it has at least 80% sequence identity to SEQ ID NO: 2, is capable of degrading human complement protein C3. 2. The protein of claim 1, which is isolated from Streptococcus pneumoniae. 3. The protein of claim 1, which is a recombinant protein. 4. The protein of claim 1 having a molecular weight of about 15 kDa to about 25 kDa. 5. An isolated peptide or polypeptide comprising at least 15 consecutive amino acids from the protein of claim 1. 6. An isolated peptide or polypeptide, characterized in that it includes at least 15 consecutive amino acids in SEQ ID NO:2. 7. An isolated protein, characterized in that it comprises SEQ ID NO:2. 8. 7. The protein of claim 7, having a molecular weight of about 15 kDa to about 25 kDa. 9. The protein of claim 8, which is isolated from Streptococcus pneumoniae. 10. The protein of claim 8, which degrades human complement protein C3. 11. The protein of claim 7, which is SEQ ID NO:2. 12. An isolated protein, characterized in that it comprises from about 1st to about 58th amino acid in SEQ ID NO:2. 13. The protein of claim 12, further comprising amino acids from about 90th to about 132nd in SEQ ID NO:2. 14. An isolated protein characterized in that it comprises amino acids from about 170th to about 227th in SEQ ID NO:5. 15. The protein of claim 14, further comprising amino acids from about 258 to about 300 in SEQ ID NO:5. 16. An isolated protein for degrading human complement protein C3, characterized in that the nucleic acid encoding the protein can hybridize with SEQ ID NO: 1 or its complementary chain under highly stringent hybridization conditions. 17. An isolated protein of about 15 kDa to about 25 kDa from Streptococcus pneumoniae characterized in that it is capable of degrading human complement C3. 18. An immune system stimulating composition, characterized in that it comprises an effective amount of an immune system stimulating peptide or polypeptide having at least 15 consecutive amino acids derived from a protein that is identical to SEQ ID NO :2 have at least 80% sequence identity and are capable of degrading human complement protein C3. 19. The immune system stimulating composition of claim 18, wherein the protein is isolated from Streptococcus pneumoniae. 20. The immune system stimulating composition of claim 19, further comprising at least one other immune system stimulating protein, peptide or polypeptide isolated from Streptococcus pneumoniae. twenty one. An immune system stimulating composition, characterized in that it includes at least a part of a protein in a therapeutically effective amount, wherein the nucleic acid encoding the protein can hybridize with SEQ ID NO: 1 or its complementary chain under highly stringent hybridization conditions down hybridization. twenty two. An immune system stimulating composition, characterized in that it comprises an effective amount of at least a part of the protein as claimed in claim 17 and a pharmaceutically acceptable carrier, wherein the protein can effectively immunize or treat mammals Individuals reproduce against infection or invasion by Streptococcus pneumoniae. twenty three. The immune system stimulating composition of claim 22, wherein said protein is provided in an amount effective to provide a therapeutic effect on said mammalian subject. twenty four. An antibody characterized in that it is capable of binding to a protein having at least 80% sequence identity to SEQ ID NO: 2 and capable of degrading human complement protein C3. 25． The antibody of claim 24, which is a monoclonal antibody. 26． The antibody of claim 24, which is obtained from mouse, rat, goat, chicken, human, or rabbit. 27． An antibody capable of binding to at least a part of a protein, characterized in that the nucleic acid encoding the protein can hybridize to SEQ ID NO: 1 or its complementary chain under highly stringent hybridization conditions. 28． An isolated nucleic acid fragment is characterized in that it can hybridize with SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. 29． The nucleic acid fragment of claim 28, which is isolated from Streptococcus pneumoniae. 30． The nucleic acid fragment of claim 28, wherein the nucleic acid fragment encodes at least a portion of a protein. 31． The nucleic acid fragment of claim 30, wherein the protein degrades human complement C3. 32． The nucleic acid fragment of claim 28, which is in a nucleic acid vector. 33． The nucleic acid fragment of claim 32, wherein the vector is an expression vector capable of producing at least a portion of a protein. 34． A cell, characterized in that it comprises the nucleic acid according to claim 28. 35． The cell of claim 34, which is a bacterial or eukaryotic cell. 36． An isolated nucleic acid fragment, characterized in that it comprises about 1st to about 174th nucleotides in SEQ ID NO: 1 or its complementary strand. 37． The isolated nucleic acid fragment of claim 34, further comprising about 320th to about 492nd nucleotides in SEQ ID NO: 1 or its complementary strand. 38． An isolated nucleic acid fragment is characterized in that it comprises the nucleic acid sequence of SEQ ID NO: 1 or its complementary chain. 39． An RNA fragment, characterized in that it is transcribed by a double-stranded DNA sequence comprising SEQ ID NO: 1 or its complementary strand. 40． A method of generating an immune response against Streptococcus pneumoniae in a mammal, comprising the step of administering to the mammal a composition comprising a therapeutically effective amount of one of At least a part of the protein, wherein the nucleic acid encoding the protein can hybridize to SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. 41． The method of claim 40, wherein the immune response is a B cell response, a T cell response, an epithelial cell response or an endothelial cell response. 42． 40. The method of claim 40, wherein at least a portion of a protein is at least 15 amino acids in length. 43． The method of claim 40, wherein the composition further comprises at least one other immune system stimulating peptide, polypeptide or protein from Streptococcus pneumoniae. 44． The method of claim 40, wherein at least a portion of a protein comprises at least 15 amino acids of SEQ ID NO:2. 45． A method for inhibiting the degradation of Streptococcus pneumoniae-mediated C3, characterized in that it comprises the steps of: contacting Streptococcus pneumoniae bacteria with an antibody capable of having at least 80% sequence identity with SEQ ID NO: 2 of protein binding. 46． A method for inhibiting C3-mediated inflammatory response and xenotransplantation rejection, characterized in that it includes expressing a protein with the amino acid sequence of SEQ ID NO: 2 on the surface of an animal organ used for xenotransplantation. 47． An isolated nucleic acid molecule, characterized in that it comprises a region of at least 15 nucleotides and can hybridize to at least one region in SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions. 48． An isolated nucleic acid molecule characterized in that it comprises a sequence capable of hybridizing to at least a region of SEQ ID NO: 1 or its complementary strand under highly stringent hybridization conditions, wherein the region is selected from nucleotides 1-174 and 320-492. 49． An isolated nucleic acid molecule comprising a region of at least 15 nucleotides capable of hybridizing to at least one region in SEQ ID NO: 4 or its complementary strand under highly stringent hybridization conditions. 50． An isolated nucleic acid molecule characterized in that it comprises a sequence capable of hybridizing to at least a region of SEQ ID NO: 4 or its complementary strand under highly stringent hybridization conditions, wherein the region is selected from nucleotides 507-681 and 827-999. 51． The nucleic acid molecule of claim 49, which encodes at least a portion of a protein. 52． The nucleic acid molecule of claim 51, wherein the protein has a predicted amino acid sequence as shown in SEQ ID NO:5. 53． An isolated nucleic acid fragment, characterized in that it has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. 54． An immune system stimulating composition comprising at least a portion of a protein in a therapeutically effective amount, wherein the nucleic acid encoding the protein can hybridize to SEQ ID NO: 4 or its complementary strand under highly stringent hybridization. 55． A method of generating an immune response against Streptococcus pneumoniae in a mammal, comprising the step of administering to the mammal a composition comprising a therapeutically effective amount of one of At least a part of the protein, wherein the nucleic acid encoding the protein can hybridize to SEQ ID NO: 4 or its complementary strand under highly stringent hybridization conditions. 56． An immune system stimulating composition, characterized in that it comprises a therapeutically effective amount of at least a part of a protein as claimed in claim 51 and a pharmaceutically acceptable carrier, wherein the protein is effective for immunization or Treatment of mammals against infection or invasive propagation of Streptococcus pneumoniae. 57． The immune system stimulating composition of claim 56, wherein said protein is provided in an amount effective to provide a therapeutic effect on said mammalian subject. 58． The immune system stimulating composition of claim 56, wherein the composition is a vaccine. 59． A polypeptide, characterized in that it has SEQ ID NO: 5. 60． The immune system stimulating composition according to claim 23, wherein the protein encoded by the nucleic acid sequence or its complementary chain can inhibit the transcription or translation of at least one endogenous Streptococcus pneumoniae nucleic acid sequence. 61． The immune system stimulating composition according to claim 56, wherein the protein encoded by the nucleic acid sequence or its complementary chain can inhibit the transcription or translation of at least one endogenous Streptococcus pneumoniae nucleic acid sequence.

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