JP2001514527A - Screening methods for proline-rich proteins - Google Patents

Abstract

SUMMARY OF THE INVENTION Methods for screening pathogenic microorganisms for secreted proteins or cell surface proteins that are potentially useful as vaccine antigens include proline-rich proteins at the amino acid level or preferably at the DNA level by using PCR. The step of identifying Suitable consensus sequences are described as (PX _m ) _n P, or (PPX _q ) _n PP, where P is proline; X is any amino acid, m is 1-5; Is 1 or 2; and n is 2 or more.

Description

DETAILED DESCRIPTION OF THE INVENTION            Screening methods for proline-rich proteins Field of the invention   The present invention relates to proteins secreted either from a microorganism or on its cell surface. The present invention relates to identification of proteins and genes encoding them. These proteins Can be useful as antigens for the preparation of vaccines against microorganisms. Background of the Invention   Characterization of proteins produced by pathogenic microorganisms is, of course, well known and active. This is a new research field.   One particular area of interest is secretory proteins, export proteins, and cells The identification of surface proteins, which may be vaccines, especially subunit vaccines ( Compositions comprising one or more antigens capable of eliciting an immune response). You.   Such proteins have been characterized from the microbial genome now available This is useful if it can be easily identified from a vast amount of sequence information that is not. Sequence motifs characteristic of proteins exported or localized on bacteria in bacteria Identification is a rapid screening of the genome for potential virulence factors and vaccine components. Make learning easier.   The presence of a proline-rich region in a protein is a good indicator of its cellular localization Something has now been found. Using this information, even in the absence of sequence information Data by designing primers based on degenerate proline-rich sequences Can screen sequence information in the base and amplify DNA from microorganisms obtain. Description of the invention   According to the present invention, secretion comprising the step of identifying a proline-rich protein Screen pathogenic microorganisms for proteins or cell surface proteins A method is provided for: Preferably, the method further comprises identifying the identified Selecting a phosphorus-rich protein.   As used herein, the term “proline-rich” protein refers to More than the average proline content of the protein of the particular microorganism being screened A protein having a qualitatively high content.   The proline-rich protein preferably has the following general sequence:                         (PX_m)_nP or (PPX_q)_nPP Comprises one or more amino acid motifs having: wherein P is proline; X is M is any amino acid; m is 1 to 5 (preferably m = 1 to 3); Is 2; and n is 2 or more. (Preferably n = 6). n repeat It is understood that m and q may be the same or different for each. Understood. It is understood that the various X residues may be the same or different. .   A preferred motif (hereinafter referred to as the proline-rich region (PRR)) is Array (PX_m)₆Represented by P. PX_mValue of m for each of the six repeats of May be the same or different. Therefore, the PRR must have at least one But seven non-consecutive, separated by no more than five other amino acids Represents a stretch of proline residues. Sequence in which 20 amino acids are randomly distributed Is very low (7.8 × 10^-Ten).   These sequences show that proline residues are relatively close to each other one by one (eg, PX PXP, or PXXPXXPXXP), or by two (for example, PPXPPXPPXPP or PPXXPP XXPPXXPP), or a mixture of both (eg, PPXXPXXPP). It is understood that it represents a motif. In other words, this motif has the general sequence (P_s X_m)_nP_sWhere each s is independently 1 or 2.   Residue X in these general sequences, which can be any amino acid, is May be the same as each other, but are generally different. The upper limit for n is, of course, Depends on the protein and the microorganism in question, and It can be large for proteins (eg, more than 10).   The screening method can be applied to any microorganism, but is preferably applied to bacteria or Applies to pathogenic microorganisms such as parasites. Bacteria can be Gram positive or Gram May be negative, for example, Helicobacter pylori, Neisseria meningitidi s, Mycobacterium tuberculosis, Escherichia coli, Neisseria gonorrhoeae, Haemophilus influenzae, Bordetella pertussis, Vibrio cholerae, and Bac illus subtilis. Parasites include protozoa (eg, Leishmania, mala Rear parasite Plasmodium, or Legionella). To other suitable microorganisms Include Saccharomyces, Chlamydia, and Borrelia burgdorferi.   The screening method uses a protein whose sequence is known but whose cellular localization is not known. By identifying the quality, it can be performed at the amino acid level. This is a computer For example, in the GCG Wisconsin Package (version 8.1 or later) It can be conveniently done using the FINDPATTERN program available. Microbial a Appropriate databases for source information on amino acid sequences are available from public data sources. Base SWISSPROT.   Alternatively and preferably, the screening method comprises a proline-rich amino acid DNA was identified from a library containing a consensus sequence encoding the motif. The amplification is performed at the DNA level.   When the screening method is performed at the DNA level, the method of the present invention preferably comprises Of a microbial DNA library into DNA encoding a proline-rich amino acid sequence. And screening. Preferably, the first step comprises a professional The step of selecting a DNA encoding the phosphorus-rich amino acid sequence follows.   This library is suitably a plasmid genome prepared using known techniques. Library. For example, screening Helicobacter pylori Suitable libraries for this are described in Censini et al. (1996) PNAS USA 93: 14648-14653. And CoVacci et al. (1993) PNAS USA 90: 5791-5795.   The step of screening the DNA library can be any suitable screening DNA that encodes a proline-rich amino acid sequence Includes screening and amplification of specific DNA sequences, including sequences. Appropriate disc The leaning step includes at least one polymerase chain reaction (PCR) step I do.   In a preferable method, the screening step is performed by using a known method. The preparation of degenerate DNA primers encoding the enriched amino acid sequence. Professional Phosphorus-rich amino acid sequences can be elucidated from commonly known proteins, or Specifically, a known secreted protein or protein, generally or from a particular microorganism of interest. Is determined by determining consensus sequences from cell surface proteins Can be done.   Using a degenerate primer, the degenerate primer sequence (or hybridize to it) Amplifying a particular sequence containing one of the Generate a population of DNA. The sequence thus obtained can then be sequenced, Then, using a reverse PCR primer and an internal primer, DNA containing a sequence encoding the amino acid sequence can be obtained.   The screening process may further involve prokaryotic or eukaryotic host cells. Expressing the DNA thus obtained in a suitable host organism such as I do.   According to a further aspect of the present invention, identified by the screening method of the present invention. Provided are proline-rich proteins. Such proteins are suitable It may be formulated with pharmaceutical excipients, such as carriers, and may contain other antigens and And / or one or more adjuvants.   In particular, a protein comprising the amino acid sequence shown in FIG. 2 or FIG. 3 (SEQ ID NOs: 2 to 4) A protein or a functionally active fragment or derivative thereof is provided. This specification As used herein, the term “functionally active” refers to a substantial biological activity. Sex, preferably antigenicity. This protein is H.pylori Was identified as a proline-rich protein.   According to a further aspect of the invention, the proteins of the invention (or their functional (E.g., RNA or DNA) encoding a fragment or derivative active at ) Is provided. In particular, the protein of FIG. 2 or a functionally active fragment thereof A DNA sequence is provided which encodes the derivative or derivative. This DNA sequence is preferably , SEQ ID NO: 1 or a fragment thereof (eg, the coding sequence of SEQ ID NO: 1) Including.   According to a further aspect of the invention, a proline-rich protein of the invention or An immunogenic composition comprising a nucleic acid encoding a protein such as Lorin-rich proteins (or nucleic acids encoding such proteins) Producing such a composition comprising associating it with a biologically acceptable excipient A method is provided for doing so.   The immunogenic composition of the invention also preferably comprises one or more additional antigenic H.py. Contains lori protein. These additional proteins are proline-rich May be the protein itself, but this need not be the case, and For example, it can be VacA, CagA, or NAP.   In particular, either the protein of FIG. 2 or the nucleic acid encoding the protein of FIG. An immunogenic composition comprising: and a pharmaceutically acceptable nucleic acid comprising the protein or nucleic acid. Methods for producing such compositions comprising the step of associating with excipients are provided. Provided.   The immunogenic composition of the invention is preferably formulated as a vaccine. these A vaccine can be prophylactic or therapeutic. And preferably one or more Including adjuvant. Such vaccines are typically referred to as "pharmaceutically acceptable. Excipient "(ie, an antibody that is itself harmful to the individual receiving the composition) (Any excipient that does not induce production). Appropriate Carriers are typically large, slowly metabolized macromolecules (eg, tantalum). Protein, polysaccharide, polylactic acid, polyglycolic acid, polymeric amino acids, Amino acid copolymers, liquid aggregates (eg, oil droplets, or liposomes), and And inactivated virus particles). Such carriers are well known to those skilled in the art. . In addition, these carriers function as immunostimulants ("adjuvants"). obtain. In addition, antigens include bacterial toxoids (eg, diphtheria, tetanus, cholera). , H, pylori, etc.).   Preferred adjuvants for enhancing the efficacy of the composition include but are not limited to Not limited to: (1) Aluminum salt (alum) (for example, aluminum hydroxide (Aluminum, aluminum phosphate, aluminum sulfate, etc.): (2) oil-in-water emulsion formulation (Other specific immunostimulants (eg, the presence of muramyl peptides or bacterial cell wall components) In the presence or absence (eg, (a) microfluidiz 5% squalene, 0.5% Tween 8 formulated into sub-μm particles using er) 0, and 0.5% Span 85 (various amounts of MTP-PE as needed (see below) MF59 (W090 / 14837), including (but not required), (b) emulsions smaller than μm Microfluidized or vortexed to larger particle size emulsion 10% squalene, 0.4% Tween 80, 5% pluronic (pluro) nic) SAF containing blocked polymer L121 and thr-MDP, and (c) 2% screen Allene, 0.2% Tween80, and monophosphoryl lipid A (MPL), trehalose di One or more from the group consisting of mycolic acid (TDM) and cell wall skeleton (CWS) Bacterial cell wall components (preferably, MPL and CWS (Detox^TMRibi including))^TMHorse mackerel Luvant (RAS) (Ribi Immunochem, Hamilton, MT); (3) Saponin adjuva (For example, Stimulon^TM(Cambridge Bioscience, Worcester, MA)) Particles that can be generated or generated therefrom, such as ISCOMs (immunostimulatory complexes) (4) Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (5) cytokines (eg, interleukins (eg, IL-1) , IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg, I FN-γ), macrophage colony stimulating factor (M-SCF), tumor necrosis factor (TNF) And (6) act as an immunostimulant to enhance the efficiency of the composition Substance. Alum and MF59 are preferred.   Muramyl peptides include, but are not limited to: N-acetylmurami Le-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L- Alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-a Soglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hirodoki (Cyphosphoryloxy) -ethylamine (MTP-PE) and the like.   Immunogenic compositions (eg, antigens, pharmaceutically acceptable carriers, and adjuvants) Bunt) is typically a diluent (eg, water, saline, glycerol, ethanol). Knol). In addition, auxiliary substances, such as wetting or emulsifying agents, pH adjustment Substances and the like can be present in such vehicles.   Typically, the immunogenic composition will be a liquid solution or suspension as an injectable. As a solid; solid form suitable for dissolution in a liquid vehicle prior to injection Solid forms suitable for suspension in a liquid or liquid vehicle may also be prepared. Preparation Also adjuvants, as discussed above for pharmaceutically acceptable carriers. It can be emulsified or encapsulated in liposomes for enhanced skin effect.   An immunogenic composition used as a vaccine comprises an immunologically effective amount of Including the peptide, and optionally any of the other components mentioned above. " An "immunologically effective amount" is a series of doses, whether in a single dose, administered to an individual. It means being effective for treatment or prevention, even as a part. This amount is Health and physical condition of the individual being placed, taxonomic group of individuals being treated ( Non-human primates, primates, etc.), the ability to synthesize antibodies of the individual's immune system, Degree of protection desired, vaccine formulation, evaluation of a physician treating the medical situation, And depends on other related factors. This amount is in a relatively wide range, Can be determined by routine trials.   Immunogenic compositions are conventionally administered parenterally (e.g., subcutaneously or intramuscularly). (By injection into it). Further formulations suitable for other aspects of administration are Oral and pulmonary formulations, suppositories, and transdermal applications. Dosing treatment is a single dose It can be a schedule or a multiple dose schedule. Vaccines are not immune It may be administered with a nodal agent.   As an alternative to protein-based vaccines, DNA vaccination can be used (Eg, Robinson and Torres (1997), Seminars in Immunology 9: 271). -283; Donnelly et al. (1997) Annu Rev Immunol 15: 617-648).   According to a further aspect of the invention, an antibody specific for a protein according to the invention is provided. Provided.   According to a further aspect of the present invention there is provided a vector comprising a nucleic acid according to the present invention. Also provided is a host organism transformed with a vector according to the invention. BRIEF DESCRIPTION OF THE FIGURES   Figure 1 used to identify and prepare proline-rich H. pylori protein 1 schematically illustrates the PCR technique performed. The upper panel (A) shows the degenerate proline primer and And pBluescript^TMFirst PCR experiment using vector universal primers Put on. The lower panel (B) shows reverse plating of internal specific primers and vector. A second PCR experiment using the immer is described.   FIG. 2 shows the nucleotides of SEQ ID NO: 1 of the proline-rich H. And SEQ ID NO: 2. Putative leader peptide and proline-rich The chicon consensus sequence is underlined, and "SD" stands for Shine-Dalgarno (Sh ine-Dalgarno) sequence.   FIG. 3 shows the sequences of proline-rich proteins from H. pylori type I and type II strains. The sequences numbered 2 to 4 are compared. "----" is found in proteins from type II strains Indicates amino acids that are not used.   FIG. 4 shows a Western analysis of the H, pylori extract. The antiserum used was Evoked against proline-rich proteins. Detailed description of the embodiments   Unless stated otherwise, the practice of the present invention is based on computer search, molecular biology, microbiology, Uses conventional techniques of biology, recombinant DNA, and immunology, and Within the scope of the person. Such techniques are explained fully in the following references: Examples For example, Molecular Cloning; A Laboratory Manual, 2nd edition (Sambrook, 1989); D NA Cloning, Volume I and Volume ii (ed. By D.N Glover, 1985); Oligonucleotide Syn thesis (edited by M.J.Gait, 1984); Nucleic Acid Hybridization (B.D.Hames and S) .J.Higgins eds., 1984); Transcription and Translation (B.D. Hames and S.J.) Higgins ed., 1984); Animal Cell Culture (R. I. Freshney ed., 1986); Immobili zed Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to  Molecular Cloning (1984); the Methods in Enzymology series (Academic Pre ss, Inc.), especially Volumes 154 and 155; Gene Transfer Vectors for Mammalia n Cells (Ed. Miller and Calos, 1987, Cold Spring Harbor Laboratory); Maye r and Walker eds. (1987), Immunochemical Methods in Cell and Molecular Biol ogy (Academic Press. London); Scopes, (1987) Protein Purification; P rinciples and Practice, 2nd edition (Springer-Verlag, N.Y.), and Handboo k of Experimental Immunology, Volumes I-IV (Edited by Weir and Blackwell, 1986).1) Computer analysis of protein sequences   That the proline-rich protein has a specific function in the microorganism, and Specifically correlates with secreted / exported or cell surface proteins Was considered. Based on this, the FINDPATTERN program (GCG Wisconsin package, version 8.1) using the SWISSPROT database in the following array Screened for sequences containing:                         (PX_m)_nP or (PPX_q)_nPP here,   P is proline; X is any amino acid; m is 1-3; q is 1 Is 2; and n is 2 or more. These sequences have already been characterized Designed to detect any type of proline-rich motif in proteins did.   The search was initially limited to three microbial classes: protozoa, gram-positive cells Bacteria, and Gram-negative bacteria. The results were analyzed for cell localization and / or Classified by physical function.   These results indicate that screening for proline-rich motifs It clearly demonstrates that it produces a relevant proportion of membrane-bound proteins. In fact, Among the proteins identified in this way, there is an open against Bordetella pertussis. Two major components of the issued non-cellular vaccine, namely, hemagglutinin (FHA) ) And pertactin (Rappuoli et al. (1991) TIBTECH 9: 232-238).   These results indicate that the analysis for the presence of proline-rich amino acid sequences General principles that are useful for identifying secreted or cell surface proteins Clearly support.   Furthermore, since the present invention was first conceived, further research has been carried out. , Thereby further confirming this approach.   First, PRB released 17567 prokaryotic proteins in SWISSPROT, Release 34. R motif [(PX_1-5)₆P], version 9.0 GCG Wisconsin package Search using the FINDPATTERN program. In 190 bacterial proteins 1536 motifs were identified (ie 1% of all analyzed proteins were young). Over dry). Of these, 98 were from gram-negative bacteria and the remaining 92 Was derived from Gram-positive bacteria. Either known function or putative cell localization Based on [PSORT algorithm, http://psort.nibb.ac.jp], 68 out of 98 (69 . 4%) and 64 out of 92 (69.5%) sequences are surface-localized or exportable tampers Quality was found.   Second, Escherichia coli, Haemophilus influenzae, Helicobacter pylori , Methanobacterium thermoautothropicum, Methanococcus jannaschii, and Search the published genome of Mycoplasma pneumoniae for the presence of the PRR motif did. Of the 53 proteins identified by this method, 39 (73.6%) Putative or exportable, therefore, the PRR motif may be That are preferentially found in proteins localized to H.influenzae In all five PRR-containing proteins are surface-localized or exportable It is estimated that 2) Detailed analysis of PRR-containing proteins   Contains 29 PRRs found in the genomes of E. coli, H. pylori, and H. influenzae Proteins were analyzed in more detail (see page 11 in English and page 13 in translation). PPR is underlined).   The average length of the PRR was about 32 amino acids.   The shortest PRR was found in the RhsA transmembrane protein of E. coli (14 residues):   The longest was in the H.pylori TonB homolog Hp1341 (56 residues)   It is clear that in addition to the PRR itself, the adjacent regions are also rich in proline.   The table below shows the bias for proline residues in the PRR. Amino acid group of PRR The formation is clearly different from the average across the genome:   The distributions shown in the last three columns in the table are generally As for, but with less hydrophobic and aromatic residues appearing in the PRR However, proline is clearly present in excess. This analysis shows that the PRR is usually , Suggesting that it is present in a hydrophilic region of the protein. 3) Analysis of H.pylorl genomic library   To extend the principles demonstrated above, this microorganism was selected by choosing the microorganism H. pylori. The effectiveness of the cleaning method was demonstrated. The experiment is a new vaccine for H. pylori H. pylori type I and type II strains for use as candidate antigens in The goal was to identify and characterize surface-displaying proline-rich proteins that pass through.   Using PCR, the H. pylori type I strain CCUG 17874 (Culture Collection of the Uni proline-rich protein from the versity of Gotheborg genomic library The DNA sequence encoding was identified and amplified. The library is pBluescrlpt (S K +) contained HindIII digested genomic DNA (Censini et al., And Covacci et al., Supra). .   The library is screened for six different proline-rich pentapeptide sequences. I was leaning. Edited about H.pylori using GCG Wisconsin package According to the codon preference table, these six sequences were combined with the following degenerate 15-mer primers: Translated to   In six separate reactions, 100 ng of plasmid DNA and 50 pmol of each degenerate Primers and upstream generic pBluescript T3 primers were used for PCR amplification. (FIG. 1A).   In some of the reactions, more than one amplification product was obtained. This is more than one Suggest the presence of a proline-rich sequence. Each product obtained from this first PCR reaction The product was completely sequenced. This is because at one end the T3 primer sequence and And a sequence having a proline primer sequence at the other end. Among them, 1 Specific internal primers were designed using the two sequences.   This internal primer was used for the second PCR reaction. This time, pBluescript Combined with downstream T7 primers from origin (FIG. 1B). It has a T7 plug on one end. A primer sequence having a primer sequence and an internal primer sequence at the other end, and a proline Amplify the region across the rich sequence.   By combining the two sequences obtained in the two PCR reactions, pBluescript primer was used. The complete DNA sequence between the two. This sequence is used as an open reading frame (ORF). If the ORF is not complete within a single plasmid, Additional clones were screened using the terminal sequence.4) Further analysis of specific H. pylori proline-rich proteins   One of several proline-rich ORFs identified in this way was used for further analysis. Selected (only one) for This ORF contains 223 genes with the sequence PASAP near the C-terminus. Encodes the amino acid protein (25.5 kDa) (FIG. 2 (SEQ ID NO: 2)). 15 residues The N-terminal signal peptide indicates that this protein is a secreted protein or cell surface tag. Protein, but no legitimate promoter region was located . Secondary structure prediction analysis revealed a proline-rich motif (which folds as a loop (Which seems to be rare) resulted in a continuous α-helical structure. Hydrophobic No segment, which suggests a transmembrane region, was detected.   The protein can be formulated as an immunogenic composition and, for example, Can be used for raw.   No significant homolog was found when this sequence was first identified, Subsequent sequencing of the H. pylori genome revealed that this protein was ORF257 (Tomb et al. (1992). 7) Nature 388: 539-547). The machine about this protein No ability is described, but it has homology to conserved secreted proteins.   Using the TBLASTN algorithm, weak homology (27-31% identity) was determined by B. subti ORF6 of the lis flaA locus, ORF6 of the flagellar operon of Agrobacterium tumefaciens, And ORFB found at similar loci in Rhizobium meliliti (GenBank L76929) Could be seen.   A PCR reaction identified a protein containing the PASAP motif, but the The fact that the primers were designed for the PAPAP motif indicates that amplification Depending on the stringency used, additional proline-rich proteins We show that quality can be selected beyond these predictions.5) Presence of protein in type II strain   The presence of the CCUG 17874 (type I strain) ORF in H. pylori type II strains was determined for G21 and G50 strains. (Censini et al., (1996) supra). PCR fragment containing complete ORF High-stained HindIII digested chromosomal DNA from CCUG 17874, G21, and G50. As a probe in Southern analysis under high Bands were detected from all three strains.   The following PCR primers were designed for this ORF and used to And amplified DNA from the G50 chromosome:   The amplified DNA (925 bp) was sequenced and the results are shown in FIG. ORF type I 4 amino acid deletions at positions 197-200 and some other points between the strain and the type II strain Well preserved except for mutations.   Amplify DNA encoding amino acids 19-183 of ORF using the following PCR primers did: They also introduced EcoRI and HindIII sites (underlined). This hula Fragment into the pGEX-2T vector (Guan et al. (1991) Anal Biochem 192: 262-267). Inserted. Truncated proteins have a signal sequence and a 40 amino acid C-terminal residue. Lack. The protein is expressed as a GST fusion in E. coli and glutathione Purified using affinity chromatography. Rabbit antiserum was The recombinant protein is raised in the form of an immunogenic composition and Used in stun analysis.   34 kDa protein species detected in total protein extract from H. pylori (FIG. 4A, lane 1). Syngeneic knockout strains (generated by allelic exchange) In, no protein was detected (lane 3). Theoretical molecular weight (25kDa ) And the observed molecular weight (34 kDa) is probably due to the It may be due to high proline content. High proline content is different in SDS gels. It is known to produce constant mobility.   This antiserum also contains the H, pylori strains G27, 60190, 326, 639, 646, 6120, and And 686 (all type I strains), and 621, 650, ZU +, and ZU- (all type II strains) Reacted with a protein extract from A., but with an apparent molecular weight of 30 kDa (Figure 4). A, lanes 2, 4, 5, 6, 7). This difference in molecular weight is due to the deletion of the four residues described above. Attributable to   G27 strain culture supernatant, whole cells, and periplasm (polymyxin B) preparation The incoming protein fraction was tested by Western analysis (FIG. 4B). This tampa Proteins in whole cells (lane 1) and in the periplasmic fraction (lane 3) It was detected but not in the culture supernatant (lane 4). This is Perip Suggest localization to rasm. The band at low molecular weight is also in the cognate mutant And appear to be unrelated to the protein of interest.   The present invention has been illustrated using only examples and modifications thereto. It is understood that variations can be made while remaining within the scope and spirit of the invention. You.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/04 G01N 33/68 G01N 33/68 A61K 37/02 (72) Inventor Rappoli, Reno Italy b. ―53035 Monteligioni, Quercegrossa, Via Caramando Rei 39 (72) Inventor Aríco, Beatrice Italy ―53036 Poggibondi, Locita Vilore 10

Claims (1)

[Claims] 1. A method for screening a pathogenic microorganism for secreted or cell surface proteins, comprising identifying a proline-rich protein and selecting the proline-rich protein. 2. It comprises the amino acid motif, wherein the protein has one or more of the following general sequence, The method of claim 1: (PX _{m) n} P or (PPX _{q) n} PP Here, P is located in proline X is any amino acid; m is 1-5; q is 1 or 2: and n is 2 or more. 3. 3. The method of claim 2, wherein said m is 1-3. 4. 3. The method of claim 2, wherein n is 6. 5. The method according to any one of claims 1 to 4, comprising a step of screening a DNA library of a microorganism for DNA encoding a proline-rich amino acid sequence. 6. Screening a specific DNA sequence including a DNA sequence encoding a proline-rich amino acid sequence by PCR, and amplifying a specific DNA sequence including a DNA sequence encoding a proline-rich amino acid sequence by PCR. The method according to any one of claims 1 to 5. 7. 7. The method of claim 6, wherein said screening step comprises preparing a degenerate DNA primer encoding a proline-rich amino acid sequence. 8. A proline-rich protein identified by the screening method according to any one of claims 1 to 7. 9. 9. The proline-rich protein of claim 8, comprising the sequence of SEQ ID NO: 1, or a functionally active fragment or derivative thereof. 10. A nucleic acid encoding the proline-rich protein according to claim 8 or 9. 11. A vaccine composition comprising the protein according to claim 8 or 9, or the nucleic acid according to claim 10. 12. 12. The vaccine of claim 11, comprising associating the proline-rich protein of claim 8 or claim 9 or the nucleic acid of claim 10 with a pharmaceutically acceptable excipient. How to generate.