AU2005203729B2 - Outer surface proteins, their genes, and their use - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

APPLICANT:

Invention Title: Microscience Limited OUTER SURFACE PROTEINS, THEIR GENES, AND THEIR USE The following statement is a full description of this invention, including the best method of performing it known to me: OUTER SURFACE PROTEINS. THEIR GENES, AND THEIR USE Field of the Invention This invention relates to the identification of outer surface proteins, their genes, and their use. More particularly, it relates to their use in therapy, for immunisation and in screening for drugs.

Background to the Invention Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is the causative agent of various conditions. In particular, GBS causes: Early onset neonatal infection.

This infection usually begins in utero and causes severe septicaemia and pneumonia in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate.

Late onset neonatal infection.

This infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including osteomyelitis, septic arthritis, abscesses and -endopthalmitis.

Adult infections.

These appear to be increasingly common and occur most frequently in women who have just delivered a baby, the elderly and the immunocompromised. They are characterised by septicaemia and focal infections including osteomyelitis, septic arthritis, abscesses and endopthalmitis.

Urinary tract infections.

GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections.

Veterinary infections.

GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.

GBS infections can be treated with antibiotics.

However, immunisation is preferable. It is therefore desirable to develop an immunogen that could be used in a therapeutically-effective vaccine.

Summary of the Invention The present invention is based on the identification of a series of genes in GBS, and also related organisms, the products of which may be associated with the outer surface of the organism. and may therefore be useful as targets for immunotherapy.

According to one aspect of the invention, a peptide is encoded by an operon including any of the genes identified herein as MS4, MS10, MS11, MS14 and MS16, obtainable from Group B Streptococcus, or a homologue or functional fragment thereof. Such a peptide is suitable for therapeutic use, e.g. when isolated.

The term "functional fragments" is used herein to define a part of the gene or peptide which retains the activity of the whole gene or peptide. For example, a functional fragment of the peptide may be used as an antigenic determinant, useful in a vaccine or in the production of antibodies.

A gene fragment may be used to encode the active peptide. Alternatively, the gene fragment may have utility in gene therapy, targetting the wild-type gene in vivo to exert a therapeutic effect.

A peptide according to the present invention may comprise any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4, 6, 8, 10 and 12, or a functional fragment thereof.

Because of the extracellular or cell surface location, the peptides of the present invention may be suitable candidates for the production of therapeutically-effective vaccines against GBS. The term "therapeutically-effective" is intended to include the prophylactic effect of vaccines.

For example, a vaccine may comprise a peptide according to the invention, or the means for its expression, for the treatment of infection.

This vaccine may be administered to females either prior to or during pregnancy to protect mother and neonate against infection by GBS.

According to another aspect of the invention, the peptides or genes may be used for screening potential antimicrobial drugs or for the detection of virulence.

A further aspect of this invention is the use of any of the products identified herein, for the treatment or prevention of a condition associated with infection by a Group B Streptococcal strain.

Although the protein has been described for use in the treatment of patients, veterinary uses of the products of the invention are also considered to be within the scope of the present invention. In particular, the peptides or the vaccines may be used in the treatment of chronic mastitis, especially in cows.

Description of the Invention The present invention is described with reference to Group B Streptococcal strain M732. However, all the GBS strains and many other bacterial strains are likely to include related peptides or proteins having amino acid sequence homology with the peptide of M732. Organisms likely to contain the peptides include, but are not limited to, S. pneumoniae, S. pyogenes, S. suis, S. milleri, Group C and Group G Streptococci and Enterococci. Vaccines to each of these may be developed in the same way as described for GBS.

Preferably, the peptides that may be useful for the production of vaccines have greater than 40% sequence similarity with the peptides identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g. 95% similarity.

Having characterised a gene according to the invention, it is possible to use the gene sequence to establish homologies in other microorganisms. In this way it is possible to determine whether other microorganisms have similar outer surface products. Sequence homologies may be established by searching in existing databases, e.g.

EMBL or Genbank.

Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and homologues can be identified and may be useful in therapy. For example, the peptides or their active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response.

They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Suitable antibodies include monoclonal antibodies, or fragments thereof, including single chain fv fragments. Methods for the preparation of antibodies will be apparent to those skilled in the art.

The preparation of vaccines based on attenuated microorganisms is known to those skilled in the art.

Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B Streptococci or other related microorganisms. The preparation of vaccine formulations will be apparent to the skilled person.

More generally, and as is well known to those skilled in the art, a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors will be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type or health of the subject etc.

The products of the present invention were identified as follows: Todd-Hewitt broth was inoculated with GBS and allowed to grow overnight at 37°C. The cells were harvested by centrifugation and washed with Phosphate Buffered Saline (PBS). The cells were resuspended in an osmotic buffer Sucrose, 20mM Tris-HCl pH 7.0, 10mM MgC1 2 containing protease inhibitors (1 mM PMSF, 10 pM Iodoeacetic Acid, 10 mM 1,10-Phenanthroline, 1 AM Pepstatin A) and Mutanolysin at a final concentration of 4 Units per microlitre. This was incubated (shaking) at 37 0 C for 2 hours.

Cells and debris were removed first by high speed centrifugation, then ultra-centrifugation for 1 hour. The resultant supernatant containing cell wall proteins was concentrated under pressure using an ultrafiltration device (10,000 molecular weight cut-off).

The sample was dialysed against ultra high quality water and lyophilised. After resuspension in loading buffer, the proteins were separated by preparative 2- Dimensional-Gel Electrophoresis. Following electrophoresis an individual spot was chosen for study. The spot was subjected to in-gel tryptic digestion. The resulting peptides were extracted from the gel and purified using microbore RP-HPLC. Fractions were collected every seconds and a portion of these consistent with the regions of UV absorbance were analysed by Delayed Extraction-Matrix Assisted Laser Desorption-Time of Flight Mass Spectrometry (DE-MALDI-TOF-MS). Peptides not observed in a blank preparation were then subjected to sequencing using Nanospray-MS/MS Using this peptide sequence information, degenerate oligonucleotides were designed to be used in a polymerase chain reaction (PCR) to amplify the DNA segment lying between the peptide sequences identified.

PCR amplification resulted in the production of several polynucleotide fragments, each of which was cloned into the pCR 2.1-TOPO vector (Invitrogen BV, Netherlands) according to manufacturers protocol.

The DNA fragment in each plasmid was identified by sequencing and then used to obtain the full-length gene sequence, as follows.

Using the identified DNA fragment, oligonucleotide primers were designed for genomic DNA sequencing. These primers were designed so as to sequence in an 'outward' direction from the obtained sequence. Once read, the sequence obtained was checked to see if the 5' and 3' termini of the gene had.been reached. The presence of these features was identified by checking against homologous sequences, and for the 5' end the presence of an AUG start codon (or accepted alternative) preceded by a Shine-Dalgarno consensus sequence, and for the 3' end, the presence of a translation termination (Stop) codon.

Upon identification of the full-length gene, primers were designed for amplification of full-length product from GBS genomic DNA. Primers used included restriction enzyme recognition sites (NcoI at the 5'end and Eco0109I at the 3' end) to allow subsequent cloning of the product into the Lactococcal expression system used.

PCR was carried out using the primers, and the products cloned into a pCR 2.1 cloning vector (In Vitrogen). Following confirmation of the presence of the cloned fragment, the DNA was excised using the restriction enzymes NcoI and EcoO109I.

The vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). This vector, harbouring a lactococcal origin of replication, a chloramphenicol resistance marker, an inducible nisin promoter and a multicloning site was altered by the replacement of the multicloning site with two 10X His tags, flanked on the most end with an NcoI site, split in the middle with a multicloning site (including an EcoO109I site), and a Stop (termination) codon at the 3'end of the His tags.

The gene of interest was inserted so that a 10X His tag was in the 3' position relative to the coding region.

Following transformation of the recombinant plasmid into L.lactis (strain NZ9000 Kuipers, O. P. et al. (1998) supra), a 400 ml liquid culture was set up and translation of the protein was induced by the addition of nisin to the culture. After a 2 hour incubation, the cells were harvested and lysed by bead beating. The resultant lysate was cleared by centrifugation, then passed over a metal affinity (Talon, Clonetech) column. The column was washed repeatedly before bound proteins were eluted with Imidazole.

To identify fractions containing the His-tagged recombinant protein, an aliquot from each fraction was analysed by SDS-PAGE, Western blotted and probed with anti- His antibodies.

The recombinant protein obtained was then used to immunise New Zealand white rabbits, with pre-immune sera being harvested prior to immunisation. Following a boost, the rabbits were sacrificed and sera collected. This sera was used in Western blots, ELISA and animal protection models.

Using the sera obtained from the animal studies, immunosorption studies were carried out.

Group B Streptococcus was grown in 20ml Todd Hewitt broth (THB) for 8 hours, harvested and resuspended in PBS. 50A1 aliquots of this were used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This was left at 4 C overnight to allow for absorbance of the bacteria onto the plate. Plates were washed twice with PBS, then blocked with 3%BSA in PBS for lhr at 37°C. Plates were again washed. Serial 10 fold dilutions of the sera were made in PBS and 50Il of these dilutions were added to the wells of the plate, in duplicate. The plate was covered and incubated for 1 hr at 37°C. The plate was washed, then anti-rabbit alkaline phosphatase conjugated secondary antibody at a concentration of 1:5000 was added to each well. Following incubation at 37 0 C for an hour, the plate was washed again. 50g1 substrate (PNPP) was added to each well, and the reaction allowed to proceed for 30min before the absorbance was read at 405 nm.

Animal protection studies were also carried out to test the effectiveness of protection on the immunised rabbits.

GBS M732 was grown up in THB until mid-log phase was reached approximately 5 hours. Cells were counted in a counting chamber, and bacteria were diluted to give a concentration of 2x10 7 bacteria per ml in pre-immune or test sera. 50l of this was injected via the intraperitoneal route into 0-1 day old mice. The mice were observed for survival over 48 hours.

The following Examples illustrate the invention.

Example 1 A first plasmid was termed MS4. The cloned DNA fragment was sequenced and the nucleotide and deduced amino acid sequence (SEQ ID NO. 1 and 2) was used to search protein databases.

Homologues to the GBS MS4 gene product can be identified in Clostridium perfingens, Haemophilus influenzae, Neisseria flavescens and Thermatoga maritima.

In all cases the homologues are the genes for Ornithine -Carbamoyltransferase (OCT). In eukaryotic systems this enzyme catalyses the second step in the _Urea cycle, the conversion of ornithine to citrulline, a reaction requiring carbomyl phosphate. In prokaryotes, ODC is one of the three enzymes involved in Arginine Deaminase activity a system which protects bacteria from acid damage. In particular, ODC is responsible for the conversion of citrulline to ornithine and carbamoyl phosphate (the opposite role to that in eukaryotes) (Casiano-Colon, A and Marquis, R. E. 1988. Appl. Environ. Microbiol. 54: 1318- 1324, Cunin, R. et al. 1986. Microbiol. Rev. 50: 314-352).

Animal protection studies were carried out as described above. The results are as follows: Treatment pups pups surviving at time (hrs) 24 48 PBS 15 6 0 Pre-Immune 41 18 1 Test 41 33 14 Example 2 A second plasmid was termed MS11. The nucleotide and deduced amino acid sequence (SEQ ID NOS. 3 and 4) were used to search protein databases.

Homologues to the GBS MS11 gene product can be identified in Lactobacillus delbrueckii, Thermotoga maritima, Clostridium acetobulylicum, Bacillus megaterium, Triticum aestivium and Synechocystis PCC6803.

In all cases the homologues are the genes for the protein Phosphoglycerate Kinase (PGK). PGK is a major enzyme in the glycolytic pathway, being involved in the conversion of Glyceraldehyde-3-phosphate to Phosphoenolpyruvate. In particular, it is involved in the catalysis of the reaction between Glycerate-l,3-diphosphate and 3-Phospho-Glycerate, releasing a phosphate in the forward reaction.

Example 3 A third plasmid was termed pMS16. The 5' and 3' cloned DNA fragments were sequenced and the nucleotide and deduced amino acid sequences for each are shown as SEQ ID NOS. 5 and 6 for the 5' fragment and SEQ ID NOS. 7 and 8 for the 3' fragment.

Homologues to the GBS MS16 gene product can be identified in Bacillus stearothermophilus, Bacillus subtilis and Mycoplasma genitalium.

In all cases the homologues are the genes for the protein Glucose-6-Phosphate Isomerase (GPI).

The enzyme Glucose-6-Phospate Isomerase catalyses the reaction between Glucose-6-phosphate and Fructose-6- Phosphate in both glycolysis (G6P to F6P) and gluconeogenesis (F6P to G6P). Mutations in the gpi gene have been shown to confer purine analogue sensitivity to organisms.

Example 4 A fourth plasmid was termed pMS14. The cloned DNA fragment was sequenced and the nucleotide and deduced amino acid sequence (SEQ ID NOS. 9 and 10) was used to search protein databases.

Homologues to the. GBS MS14 gene product can be identified in Bacillus subtilis, Bacillus stearothermophilus, Mus musculus, Bos taurus and Zea mays.

In all cases the homologues are the genes for the protein Purine Nucleoside Phosphatase (PNP). The function of this enzyme is to cleave the nucleosides guanosine or inosine to their respective basis and sugar-1-phosphate molecules in the presence of orthophosphate.

Example A fifth plasmid was termed pMS10. The cloned DNA fragment was sequenced. The nucleotide and deduced amino acid sequence (SEQ ID NOS. 11 and 12) was used to search protein databases.

Homologues to the GBS MS10 gene product can be identified in Streptococcus mutans, Nicotiana plumb, Pisum sativum and Zea mays. In all cases the homologues are the genes for the protein Nonphosphorylating, NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase (NPGAP-3-DH).

NPGAP-3-DH has been reported as being an important means of generating NADPH for biosynthetic reactions in S. mutans (as opposed to NAD-specific GAP-3-DH which satisfies the requirements of the glycolytic pathway) (Boyd, D.A., Cvitkovitch, D. G. and Hamilton, I. R 1995 J. Bacteriol.

177: 2622-2727).

Claims (7)

1. 4. A vaccine comprising a peptide according to claim 1, or the means for its expression. Use of a product according to any of claims 1 to 3, for screening potential drugs or for the detection of virulence.
2. 6. Use of a product according to any of claims 1 to 3, for the manufacture of a medicament for use in the treatment or prevention of a condition associated with bacterial infection.
3. 7. Use according to claim 6, wherein the infection is a Group B streptococcal infection.
4. 8. Use according to claim 6 or claim 7, wherein the infection is a focal infection.
5. 9. Use according to claim 6 or claim 7, wherein the infection is a urinary tract infection. An antibody raised against a peptide according to claim 1. SEQUENCE LISTING <110> MicroscienCe Limited <120> OUTER SURFACE PROTEINS, THEIR GENES, AND THEIR USE <130> REP05969W0 <140> <141> <160> 12 <170> Pateritln Ver. 2.1 <210> 1 <211> 1014 <212> DNA <213> group B streptococcus <220> <221> CDS, <222> (1014) <400> 1 atg aca Met Thr 1 caa gta ttt Gin Val Phe 5 caa gga cgt agt Gin Gly Arg Ser tta gca gaa aaa Leu Ala Glu Lys gat ttt Asp Phe tct Ser cgt gag gaa Axrg Glu Glu ttt gaa tat ctt Phe Glu Tyr Leu a tt Ile 25 gat ttt tca gct Asp Phe Ser Ala cat tta aaa His Leu Lys ggt aaa aat Gly Lys Asn gac ctt Asp Leu att gct Ile Ala aaa Lys aaa cgt ggt gtt Lys Arg Gly Val cct Pro 40 cat cat tat ctt His His Tyr Leu gaa Glu ctc tta ttt gaa Leu Leu Phe Glu aca tct act cgt Thr Ser Thr Arg act Thr cgc gca gcc ttt Arg Ala Ala Phe 144 192 240 288 act gca gca att Thr Ala Ala Ilie cta ggc gct cat Leu Gly Ala His ccg Pro gaa tac ctt ggt Glu Tyr Leu Gly gca A-l a aat gat att caa Asn Asp Ile Gin ctt Leu ggt aaa aaa gaa Gly Lys Lys Glu tca Ser 90 aca gaa gat act Thr Glu Asp Thr gct aag Ala Lys gtt. tta gga Val Leu Gly cgt Arg 100 atg ttt gat ggt Met Phe Asp Gly att Ile 105 gaa ttc cgt ggt Glu Phe Arg Gly ttt ago caa Phe Ser Gin 110 gtc tgg aat Val Trp Asn aga. atg Arg Met ggt tta. Gly Leu 130 gtt Val 115 gaa gag ctt gct Giu Glu Leu lia gaa Glu 120 ttt tot gga gta Phe Ser Gly Val. cot Pro 125 aca gat gaa tgg Thr Asp Giu Trp, cat His 135 cca aca caa atg Pro Thr Gin Met cta Leu 140 got gac tac ott Ala Asp Tyr Leu 384 432 480 528 act Thr 145 ato aaa gaa aao Ile Lys Giu Asn ggt aaa ott gaa. Gly Lys Leu Giu g gt Gi y 155 att act ott gtt Ile Thr Leu Val tgt ggt gao gga. Cys Gly Asp Gly aac aat gtt gcc Asn Asn Val Ala aac Asn 170 tog ott tta gtg Ser Leu Leu Val got ggg Al1a Gly 175 act ttg at~g Thr Leu Met cow gct gaa Xaa Ala Glu 195 ggg Gi y 180 gtc aat gia cac Val Asn Val His atc Ile 185 ttt. tot oca aaa Phe Ser Pro Lys gaa ott tty Giu Leu Phe 190 aaa gaa. tct Lys Glu Ser 576 624 gag att gtt aaa Glu Ile Val Lys ttg Leu 200 got gaa. gga tat Ala Giu Gly Tyr goo Al a 205 ggg got Gly Ala 210 cac gtt oto gtt His Val Leu Val gat aat gta gao Asp Asn Val Asp gaa Giu 220 got gta aag gga Ala Val Lys Gly gca Al a 225 gao gto ttt tao Asp Val Phe Tyr act Th~r 230 gat gto tgg gta Asp Val Trp Val tog Ser 235 atg gga gaa gaa Met Gly Giu Glu gat Asp 240 672 720 768 aag ttc aaa gaa Lys Phe Lys Glu gtt gaa ott ott Val Giu Leu Leu caa Gln 250 oca tat caa gta Pro Tyr Gin Vai aac atg Asn Met 255 gaa otg att Glu Leu Ile tta cot gca Leu Pro Ala 275 aaa Lys 260 aaa got aat aat Lys Ala Asn Asn gat Asp 265 aat ott ato tto Asn Leu Ile Phe tta cac tgo Leu His Cys 270 gao gto. got Asp Val Ala 816 864 tto cat gat aca Phe His Asp Thr aat Asn 280 aco gtt tat ggc Thr Vai Tyr Gly gaa aaa Glu Lys 290 ttt ggg gtc aag Phe Gly Val Lys atg gaa gtt act Met Giu Val Thr gaa gtc ttc cgt Glu Val Phe Arg 912 agc Ser 305 aaa tat gct cgt Lys Tyr Ala Arg cat HiS 310 ttc gac caa gct Phe Asp Gin Ala gaa Glu 315 aat cgt atg cac Asn Arg Met His 960 1008 att aaa gct gta Ile Lys Ala Val atg Met 325 gct gca acc ctt Ala Ala Thr Leu gga Gly 330 aat ctt ttc att Asn Leu Phe Ile cca aaa Pro Lys 335 1014 gtt taa Val <210> 2 <211> 337 <212> PRT <213> group B streptococcus <400> 2 Met Thr 1 Gin Val Phe 5 Gin Giy Arg Ser Leu Ala Giu Lys Asp Phe Leu Lys Ser Arg Glu Asp Leu Lys Glu Phe Giu Tyr Leu Ile 25 Asp Phe Ser Ala His Lys Arg Gly Val His His Tyr Leu Glu Gly Lys Asn Ile Ala Leu Leu Phe Glu Lys 55 Thr Ser Thr Arg Thr Arg Ala Ala Phe Thr Thr Ala Ala Ile Asp 70 Leu Gly Ala His Pro 75 Glu Tyr Leu Gly Ala Asn Asp Ile Gin Leu Gly Lys Lys Glu Thr Giu Asp Thr Ala Lys Val Leu Gly Arg Met Val 115 Arg 100 Met Phe Asp Gly Ile 105 Glu Phe Arg Gly Phe Ser Gin 110 Val Trp Asn Glu Gu Leu Ala Glu 120 Phe Ser Gly Val Pro 125 Gly Leu Thr Asp Giu Trp His Pro Thr Gin Met Leu Ala Asp Tyr Leu 140 Thr 145 Ile Lys Glu Asn Gly Lys Leu Glu Gi y 155 Ile Thr Leu Val cys Gly Asp Gly Arg 165 Asn Asn Val Ala As n 170 Ser Leu Leu Val Ala Gly 175 Thr Leu Met Xaa Ala Giu 195 Gi y 180 Val Asri Val His Ile 185 Phe Ser Pro Lys Giu Leu Phe 190 Lys Giu Ser Giu Ile Val Lys Leu 200 Ala Giu Gly Tyr Al a 205 Giy Ala 210 His Val Leu Val Thr 215 Asp Asn Val Asp Gi u 220 Ala Val Lys Gly Ala 225 Lys Asp Val Phe Tyr Phe Lys Giu Arg 245 Asp Val Trp Val Ser 235 Met Gly Giu Giu Asp 240 Val Giu Leu Leu Gin 250 Pro Tyr Gin Val Asn Met 255 Giu Leu Ile Leu Pro Ala 275 Lys 260 Lys Ala Asn Asn Asp 265 Asn Leu Ile Phe Leu His Cys 27 0 Asp Val Ala Phe His Asp Thr Thr Val Tyr Gly Lys 285 Giu Ser 305 Lys 290 Phe Gly Val Lys Giu 295 Met Glu Vai Thr Asp 300 Giu Vai Phe Arg Lys Tyr Ala Arg His 310 Phe Asp Gin Ala Glu 315 Asn Arg Met His Thr 320 Ile Lys Ala Vai Met 325 Ala Ala Thr Leu Gi y 330 Asn Leu Phe Ile Pro Lys 335 <210> <211> <212> <213> <220> 3 1197 DNA group B streptococcus <221> CDS <222> (1197) <400> 3 atg get Met Ala 1 aaa ttg Lys Leu gtt aaa gac Val Lys Asp gtt gat Val Asp 10 ttg aag gta aaa Leu Lys Val Lys aaa gte Lys Val etc gtt cgt Leu Val A-rg aac gac aac Asn Asp Asfl gaa caa ggt Glu Gin Gly gtt Val gac ttt aat gtg Asp Phe Asn Val ect Pro 25 ttg aaa gac Leu Lys Asp cgt ate act geg Arg Ile Thr Ala get Al a 40 ctt-eca aca ate Leu Pro Thr Ile gge gtt ate act Gly Val Ile Thr aag tat ate ate Lys Tyr Ile Ile gga cgt gtt aaa Gly Arg Vai Lys ggt egi get Gly Arg Ala ate Ile 55 etc tte tet cac Leu Phe Ser His ctt Leu gaa Giu gaa get gac aaa Giu Ala Asp Lys gaa Glu 70 gga aaa tea ctt Gly Lys Ser Leu ceg gta get get Pro Val Ala Ala ga t Asp 240 288 tta get get aaa Leu Ala Ala Lys ctt Leu ggt caa gat gtt Gly Gin Asp Val ttc eca ggt gtt Phe Pro Gly Val act cgt Thr Arg ggt gca aaa Gly Ala Lys ctt ttg gtt Leu Leu Val 115 tta Leu 100 gaa gaa gca ate Glu Glu Ala Ile aat Asn 105 get ttg gaa gat Ala Leu Giu Asp gga eaa gtt Gly Gin Val 110 aag aaa gaa Lys Lys Giu gaa aac act cgt Giu Asn Thr Arg ttt Phe 120 gaa gat gtt gac Giu Asp Val Asp ggt Giy 125 tet aag Ser Lys 130 aat gac gaa gaa Asn Asp Giu Glu ctt Leu 135 ggt aaa tac tgg Giy Lys Tyr Trp get Ala 140 tca ett gga gat Ser Leu Gly Asp gga Gly 145 tea Ser ate ttc gtt aac Ile Phe Vai Asn aac gta ggt att Asn Val Gly Ilie 165 gat Asp 150 gca ttt ggt aca Ala Phe Giy Thr cac egt get cat His Arg A-la His gca Al a 160 432 480 528 tea gca aac gtt Ser Ala Asn Val gaa Giu 170 aaa get gta get Lys Ala Val Ala ggt tte Gly Phe 175 ctt ctt gaa Leu Leu Giu gaa cgc cca Giu Arg Pro 195 aac Asn 180 gaa att gct tac Giu Ile Ala Tyr a tc Ile 185 caa gaa gca gtt Gin Giu Ala Vai gaa act cca Giu Thr Pro 190 tct gat aag Ser Asp Lys ttc gta get att Phe Val Ala Ile ggt ggc tca aaa gtt Giy Giy Ser Lys Val 205 att ggt Ile Giy 210 gtt atc gaa aac Val Ile Giu Asn ctt Leu 215 ctt gaa aaa get Leu Glu Lys Ala gat Asp 220 aaa gtt ctt atc Lys Val Leu Ile ggt Gly 225 ggt ggt atg act Gly Gly Met Thr tac Tyr 230 aca ttc tac aaa. Thr Phe Tyr Lys get Al a 235 caa ggt atc gaa. Gin Gly Ile Giu atc Ile 240 672 720 768 ggt aac tea ctt Gly Asn Ser Leu gta Val 245 gaa. gaa gac aaa Giu Giu Asp Lys gat gtt gct aaa Asp Vai Ala Lys gac etc Asp Leu 255 ctt gaa aaa Leu Giu. Lys gca aae gca. Al1a Asn Ala 275 tca Ser 260 aac ggt aaa ttg Asn Giy Lys Leu ate Ile 265 ttg cea. gtt gac Leu Pro Val Asp tea aaa gaa Ser Lys Giu 270 gaa ggt gaa Giu Gly Giu 816 864 ttt get ggt tat Phe Ala Giy Tyr act Thr 280 gaa gtt ege gac Giu Vai Arg Asp act Thr 285 gca gtt Ala Val 290 tea gaa ggg ttc Ser Giu Gly Phe ett Leu 295 ggt ctt gac ate Gly Leu Asp Ile ggt Gi y 300 cet aaa tea. ate Pro Lys Ser Ile get Ala 305 aaa. ttt gat gaa Lys Phe Asp Giu gca Al a 310 ett act ggt get Leu Thr Gly Ala aaa Lys 315 aca gtt gta tgg Thr Val Val Trp 912 960 1008 gga ect atg ggt Gly Pro Met Gly ggt gta atg gac Gly Val Met Asp 340 ttt gaa aae cet Phe Glu Asn Pro gac Asp 330 tte eaa. get ggt Phe Gin Ala Gly aca ate Thr Ile 335 get ate gtt aaa Ala Ile Val Lys caa Gin 345 eca. ggc gtt aaa Pro Giy Val Lys tea ate ate Ser Ile Ile 350 1056 ggt ggt ggt Gly Giy Giy 355 gat tea gca. gea Asp Ser Ala Ala get ate aae ctt Ala Ile Asn Leu ggt Gi y 365 cgt Arg get gac Ala Asp 1104 aaa ttc tca Lys Phe Ser 370 gaa ggt aaa Glu Gly Lys 385 <210> 4 <211> 398 <212> PRT <213> group tgg atc tct act Trp Ile Ser Thr 375 gta tta cca ggt Val Leu Pro Gly 390 ggt ggt gga gca Gly Gly Gly Ala atg gaa ttg ctc Met Glu Leu Leu 1152 ttg gca gca Leu Ala Ala ttg Leu 395 act gaa aaa taa Thr Giu Lys 1197 B streptococcus <400> 4 Met Ala Lys Leu 1 Thr Val Lys Asp Val Asp Leu Lys Val Lys Lys Val Leu Val Arg Asn Asp Asn Val Asp Phe Asn Val Pro 25 Leu Lys Asp Gly Val Ile Thr Tyr Ile Ile Arg Ile Thr Ala Ala 40 Leu Pro Thr Ile Lys Glu Gin Gly Gly Arg Ala Ile 55 Leu Phe Ser His Leu Gly Arg Val Lys Glu Ala Asp Lys Glu Gly Lys Ser Leu Ala 75 Pro Val Ala Ala Asp Leu Ala Ala Lys Leu Gly Gin Asp Val Phe Pro Giy Val Thr Arg Gly Ala Lys Leu Leu Val 115 Leu 100 Glu Giu Ala Ile Asn 105 Ala Leu Giu Asp Gly Gin Val 110 Lys Lys Glu Glu Asn Thr Arg Glu Asp Val Asp Gly 125 Ser Lys 130 Asn Asp Giu Glu Leu 135 Gly Lys Tyr Trp Ala Ser Leu Giy Asp 140 Gly 145 Ile Phe Val Asn Asp 150 Ala Phe Gly Thr Ala 155 His Arg Ala His Ala 160 Ser Asn Vai Gly Ile 165 Ser Ala Asn Val Glu 170 Lys Ala Val Ala Gly Phe 175 Leu Leu Giu Asn Giu Ile Aia Tyr Ile Gin Giu Aia Vai Giu Thr Pro 180 185 Giy Giy Ser Lys Ser Asp Lys Glu Arg Pro 195 Phe Vai Ala Ile Leu 200 Val 205 Ile Giy 210 Val Ile Giu Asn Leu 215 Leu Giu Lys Ala Asp 220 Lys Val Leu Ile Gly 225 Giy Gly Met Thr Tyr 230 Thr Phe Tyr Lys Al a 235 Gin Giy Ile Giu Ile 240 Gly Asn Ser Leu Val 245 Giu Giu Asp Lys Leu 250 Asp Val Ala Lys Asp Leu 255 Leu Giu Lys Ala Asn Ala 275 Ser 260 Asn Gly Lys Leu Ile 265 Leu Pro Vai Asp Ser Lys Giu 270 Glu Giy Giu Phe Ala Gly Tyr Thr 280 Giu Vai Arg Asp Thr 285 Ala Val 290 Ser Giu Gly Phe Giy Leu Asp Ile Giy 300 Pro Lys Ser Ile Ala 305 Lys Phe Asp Giu Al a 310 Leu Thr Giy Ala Lys 315 Thr Vai Val Trp Asn 320 Gly Pro Met Gly Val 325 Phe Giu Asn Pro Phe Gin Ala Gly Thr Ile 335 Giy Val Met Gly Gly Giy 355 Asp 340 Ala Ile Val Lys Gin 345 Pro Gly Val Lys Ser Ile Ile 350 Arg Ala Asp Asp Ser Ala Ala Ala 360 Ala Ile Asn Leu Gi y 365 Lys Phe 3*70 Ser Trp Ile Ser Lys Vai Leu Pro 390 Thr 375 Giy Gly Giy Ala Ser 380 Met Giu Leu Leu Gilu 385 Gly Giy Leu Ala Ala Leu Thr Giu Lys 395 <210> <211> 516 <212> DNA <213> group B streptococcus <220> <221> CDS <222> (1) .(516) <400> atg aca Met Thr 1 cat ati aca itt gac tta ttc His Ile Thr Phe Asp Leu Phe 5 gtc ttg ggt caa Val Leu Gly Gin ttt gta Phe Val gca gat Al a Asp ggc gaa cac Gly Giu His gct ttc ctt Ala Phe Leu gag Glu tia gac tac cia Leu Asp Tyr Leu cca Pro
6. 25- cca caa gia agt Pro Gin Val Ser gca Al a cgt caa ggg act Arg Gin Giy Thr ggi Gi y 40 cci gga ica gat ttt ctc gga tgg Pro Gly Ser Asp Phe Leu Gly Trp aig gaa Met Giu cci cca gaa aac Pro Pro Giu Asn gac aaa gaa gaa Asp Lys Giu Giu ict cgc ati caa Ser Arg Ile Gin gcc gct. gaa aag Ala Ala Giu Lys a ii Ile aaa ica gai agc Lys Ser Asp Ser gaa Glu 75 gia cic gig gt Val Leu Val Val ati Ile 192 240 288 ggi aii ggt ggi Giy Ile Giy Gly icg Ser iac cit ggt gca Tyr Leu Gly Ala gca gca ati gac Ala Ala Ile Asp iii tig Phe Leu aai aai cai Asn Asn His cag ati cti Gin Ile Leu 115 iii Phe 100 gci aai itg caa Ala Asn Leu Gin acc Thr 105 gca gaa gaa cgi Ala Giu Giu Arg aaa gcg cct Lys Ala Pro 110 cit gcc gat Leu Ala Asp tat gci gga aai Tyr Ala Gly Asn ict Ser 120 ati ica ici act Ile Ser Ser Thr ita gt Leu Val 130 gaa tac gtc caa Giu Tyr Val Gin gat Asp 135 aaa gaa tic ica Lys Giu Phe Ser gia Vai 140 aai gic ait ica Asn Val Ile Ser 432 480 aaa Lys 145 ica ggi aca aca Ser Giy Thr Thr act Thr 150 gaa cca gcg att. Glu Pro Ala Ile gci Al a 155 tic cgi gta iii Phe Arg Vai Phe aaa Lys 160 gaa cii cta git aaa aag iac Giu Leu Leu Val Lys Lys Tyr cgg ica aga aga agc 516 Arg Ser Arg Arg Ser <210> 6 <211> 172 <212> PRT <213> group B streptococcus <400> 6 Met Thr 1 His Ile Thr Phe Asp Leu Phe Lys Val Leu Gly Gin Phe Vai Gly GXU His Ala Phe LeU Gi u Leu Asp Tyr Leu Pro 25 Pro Gin Val Ser Ala Ala Asp Leu Gly Trp Arg Gin Gly Thr Gi y 40 Pro Gly Ser Asp Phe Met G1u Pro Pro Glu Asn Asp Lys Glu GiU Ser Arg Ile Gin Lys Ala Ala Giu Lys Ile 70 Lys Sex Asp Ser Val Leu Val Val Ile Leu Gly Ile Giy Giy Ser Tyr Leu Gly Ala Lys 90 Ala Ala Ile Asp Phe Asn Asn His Gin Ile Leu 115 Phe 100 Ala Asn Leu Gin Thr 105 Ala Giu Giu Arg Lys Ala Pro 110 Leu Al1a Asp Tyr Ala Gly Asn Ser 120 Ile Ser Ser Thr Tyr 125 Leu Val 130 Giu Tyr Vai Gin Asp 135 Lys Glu Phe Ser Asn Val Ile Ser Lys 145 Ser Giy Thr Thr Thr 150 Giu Pro Ala Ile Ala Phe Arg Vai Phe 155 Lys 160 Giu Leu Leu Val Lys 165 Lys Tyr Arg Ser Arg Arg Ser 170 <210> 7 <211> 318 <212> DNA <213> group B streptococcus <220> <221> CDS <222> (1) .(318) <400> 7 att aac cga aga ttt aga tgg tct tgg tta tct tca aga aaa gat gta Ile Asn Arg Arg Phe Arg Trp Ser Trp Leu Ser Ser Arg Lys Asp Val ga t Asp ttt gtt aat Phe Val Asn aaa aaa gca aca Lys Lys Ala Thr gat Asp 25 ggt gtg ctt ctt Giy Val Leu Leu gct cat aca Ala His Thr caa gac gct Gin Asp Ala gat ggt ggg Asp Gly Gly gtt cca aat atg Val Pro Asn Met ttt Phe 40 gta acg ctt cct Val Thr Leu Pro aca Thr tac act Tyr Thr ctt ggt tac act Leu Giy Tyr Thr att Ile 55 tac ttc ttt gag Tyr Phe Phe Giu tta LeU gca att ggc ctt Ala Ile Gly Leu tca Ser ggt tat ctt aac Gly Tyr Leu Asn tca 5cr 70 gta aat cca ttt Val Asn Pro Phe caa ccg ggg gta Gin Pro Giy Vai gaa Giu 192 240 288 gca tat aaa cgt Ala Tyr Lys Arg aat Asn atg ttc gca ttt Met Phe Ala Phe ggt Giy 90 aaa cct gga ttc Lys Pro Gly Phe gaa gag Giu Glu ctt agc gct Leu Ser Ala gaa Giu 100 ttg aat gca cgt Leu Asn Ala Arg ctt taa 318 Leu 105 <210> 8 <211> 105 <212> PRT <213> group <400> 8 Ile Asn Arg 1 Asp Phe Val. B streptococcus Arg Phe Arg Trp Ser Trp 5 Asn Lys Lys Ala Thr Asp 25 Leu 10 Ser Ser Arg Lys Asp Val Gly Vai Leu Leu Ala His Thr Asp Gly Giy Val Pro Asn Met Phe Val Thr Leu Pro Thr Gin Asp Ala Ala Ile Gly Leu Tyr Thr Leu Gly Tyr Thr Ile Tyr Phe Phe Giu Ser Gly Tyr Leu Asn Ser 70 Val Asri Pro Phe Asp Gin Pro Gly Vai Ala Tyr Lys Arg Asn Met Phe Ala Phe Giy 90 Lys Pro Gly Phe Giu Glu Leu Ser Ala Glu 100 Leu Asn Ala Arg Leu
7. 105. <210> 9 <211> 804 <212> DNA <213> group B streptococcus <220> <221> CDS <222> (804) <400> 9 atg aca Met Thr 1- tta tta gaa Leu Leu Giu 5 aaa att aat gag Lys Ile Asn Giu act Th r 10 aga gac ttt. ttg Arg Asp Phe Leu caa gca Gin Ala aaa ggc gtc Lys Giy Val gga gaa ttg Gly Giu Leu aca Thr gca cca gaa ttt Ala Pro Giu Phe ggy Xaa 25 ctt att tta Leu Ile Leu gct gaa gaa atc Ala Giu Giu Ile aat cct att gtt Asn Pro Ile Val ggc tct ggt tia Gly Ser Gly Leu gtg gat tat gca Val Asp Tyr Ala cat gct gga aaa His Ala Gly Lys 96 144 gac atc Asp Ile ccm aat tgg gga Xaa Asn Trp Gly cag Gin tca aca gta gtt Ser Thr Val Val 192 240 ttt Phe agt gta tgg gat Ser Val Trp Asp tta Leu 70 tca ggc cgt aag Ser Gly Arg Lys gta Val 75 tta gcg ctt caa Leu Ala Leu Gln ggt Gi y cgt ttt cat ttt tay gaa Arg Phe His Phe Tyr Giu ggw aat aca atg gaa gtc gtt act ttc cca Xaa Asn Thr Met Giu Val Vai Thr Phe Pro 288 gta cgt atc Val Arg Ile gca gcg ggt Ala Ala Gly 115 aga gca ttg gct Arg Ala Leu Ala tgc Cys 105 cac agt gtg ctt His Ser Val Leu gtg act aat Val. Thr Asn 110 ctg atc aaa Leu Ile Lys ggg att gga tac Gly Ile Gly Tyr gga Gi y 120 cca gga act tta Pro Giy Thr Leu atg Met 125 gac cac Asp His 130 atc aat atg att. Ile Asn Met Ile 9gg Gi y 135 act aac cct ctc Thr Asn Pro Leu ata Ile 140 ggt gag aac ctt Gly Giu Asn Leu gaa ttt gga cca Giu Phe Gly Pro ttc cca gac atg Phe Pro Asp Met tcg Ser 155 gat gct tay aca Asp Ala Tyr Thr 432 480 528 aca tat cga caa Thr Tyr Arg Gin aaa Lys 165 gct cac caa att. Ala His Gin Ile gct Al1 a 170 gaa aac gat atc Giu Asn Asp Ile aaa ctc Lys Leu 175 gaa gaa ggt Glu Giu Giy gca gaa att. Ala Giu Ile tac ttg ggt gta Tyr Leu Giy Val tca Ser 185 gga ccc act tat Gly Pro Thr Tyr gaa aca cct Giu Thr Pro 190 gta ggt atg Val Giy Met 576 624 cgt gca ttc caa Arg Ala Phe Gin aca Thr 200 atg ggc gca caa Met Giy Ala Gin gcg Al a 205 tcc acg Ser Thr 210 gtt. cca gag gtg Val Pro Giu Val atc Ile 215 gtt gca gct cac Val Ala Ala His tca Ser 220 ggg ctt aaa gtg Giy Leu Lys Val tta Leu 225 gga att tca gca Gly Ile Ser Ala att Ile 230 act aac ctt gcc Thr Asn Leu Ala gct Al a 235 ggc ttC caa tca Giy Phe Gin Ser gag Gi u 240 672 720 768 ctc aat cat gag Leu Asn His Giu gag Giu 245 gtc. gtt gaa gtt Val Val Giu Val act Thr 250 cag cgt att aaa Gin Arg Ile Lys gaa gat Giu Asp 255 ttc aag gga Phe Lys Gly tta Leu 260 ggt aaa tca tta Gly Lys Ser Leu gtt Val 265 gct gaa ctc Ala Giu Leu <210> <211> 268 <212> PRT <213> group B streptococcus <400> Met Thr Leu Leu, Glu Lys Ile Asn Glu Thr Arg Asp Phe Leu Gin Ala Lys Gly Val Gly Giu Leu Thr Ala Pro Glu Phe Xaa 25 Leu Ile Leu Gly Ser Gly Leu Asp Tyr Ala Ala Glu Glu Ile Asn Pro Ile Val Val Asp Ile Xaa Asn Trp, Gly Gin Ser Thr Val Val His Ala Gly Lys Phe Ser Val Trp Asp Leu 70 Ser Gly Arg Lys Val Leu Ala Leu Gin Arg Phe His Phe Tyr Giu Xaa Asn Thr Giu Val Val Thr Phe Pro Val Arg Ile Ala Ala Gly 115 Arg Al1a Leu Ala Cys 105 His Ser Val 1,eu Val Thr Asn 110 Leu Ile Lys Gly Ile Gly Tyr Gi y 120 Pro Gly Thr Leu Met 125 Asp Glu 145 His 130 Ile Asn Met Ile G1 y 135 Thr Asn Pro Leu Ile Gily Giu Asn 140 Asp Ala Tyr Thr Leu Ala Giu Phe Gly Pro Tyr Arg Gin Lys 165 Arg 150 Phe Pro Asp Met Ser 155 160 Lys Leu 175 Thr Ala His Gin Ile Al a 170 Glu Asn Asp Ile Giu Glu Gly Ala Glu Ile 195 Tyr Leu Giy Vai Ser 185 Gly Pro Thr Tyr Glu Thr Pro 190 *Vai Gly Met Arg Ala Phe Gin Thr 200 Met Giy Ala Gin Al a 205 Ser Thr 210 Val Pro Giu Val Val Ala Ala His Ser 220 Gly Leu Lys Val Leu Giy Ile Ser Ala Ile Thr Asn Leu Ala Ala Gly Phe Gin Ser Giu 230 23524 240 Leu Asn His Phe Lys Gay Giu Glu Val Val Glu 245 Leu Gly Lys Ser Leu 260 Val. Thr Gin Arg Ile Lys Glu Asp 250 255 Val 265 Ala Glu Leu <210> <211> <212> <213> 11 1428 DNA group B streptococcus <220> <221> CDS <222> (1)..(1428) <400> 11 tig aca Leu Thr ~aaa gaa tat Lys Giu Tyr caa aat tat gtc Gin Asn Tyr Val aat Asn 10 ggc gaa tgg aaa Gly Glu Trp LYS tca ict Ser Ser gtt aat cag Val Asn Gin ttc gtg cca Phe Val Pro att Ile gag ait ttg tca Giu Ile Leu Ser cca Pro 25 att gat gat tct Ile Asp Asp Ser tca ttg gga Ser Leu Gly atg aaa gcg Met Lys Ala gcg aig act cga Ala Met Thr Arg gaa Giu 40 gaa git gat cat Glu Val Asp His gct Ala ggt cgt Gly Arg gag gct tta cca Giu Ala Leu Pro gct Ala 55 tgg gct gct ita Trp Ala Ala Leu aca Thr gta tat gaa egi Vai Tyr Giu Arg gca Ala caa tac cit cat Gin Tyr Leu His gcc gca gac ati Ala Ala Asp Ile att Ile 75 gaa cgt gat aaa Glu Arg Asp Lys gaa Giu 192 240 288 336 gaa ait gct act Giu Ile Ala Thr gt Val tia gca aaa gaa Leu Ala Lys Giu ati Ile 90 tct aaa gct tac Ser Lys Ala Tyr aat gct Asn Ala tca gia act Ser Val Thr gag Glu 100 gtt gia agg aca Val Val Arg Thr gct gat Ala Asp 105 cit ati cgt tat gca gca Leu Ile Arg Tyr Ala Ala 110 gaa gaa gga Giu Giu Gly 115 att cgt tta tca Ile Arg Leu Ser act Thr 120 tca gct gac gaa Ser Ala Asp Giu ggi 125 gga aaa atg Gly Lys Met caa cca gta Gin Pro Val gat gct Asp Ala 130 tca aca ggt cat Ser Thr Gly His aag Lys 135 ttg got gtt att Leu Ala Val Ile cgt cgt Arg Arg 140 ggt Gi y 145 atc gtt tta gca Ile Val Leu Al1a atc Ile 150 gca cct tat aat Ala Pro Tyr Asn cct gtt aac ctc Pro Val Asn Leu tca Ser 160 432 480 528 576 gga tca aaa att Gly Ser Lys Ile cca gct. ota att Pro Al1a Leu Ile ggt Gi y 1*70 gga aao gtt gtg Gly Asn Val. Val atg ttt Met Phe 1,75 aaa got. Lys Ala aaa cca cca Lys Pro Pro ttt goa gaa Phe Ala Giu 195 aca Thr 180 caa ggt tca, gic Gin Gly Ser Val toa Ser 185 gga ctt gtt. tta. Gly Leu Val Leu gca Al a 190 gca ggt ctt oca. Ala Gly Leu Pro gca Ala 200 ggt gtc ttt aat Gly Val Phe Asn act Thr 205 att. aca gga Ile Thr Gly cgo ggt Arg Gly 210 tot gag att gga Ser Giu Ile Gly gat Asp 215 tac att gtt gag Tyr Ile Val Giu cat His 220 gaa gaa gtt aat Giu Glu Val Asn 672 720 tt Phe 225 att, aac ttt aca Ile Asn Phe Thr gga Gi y 230 toa aeg oca gtt Ser Thr Pro Val caa cgt att ggt Gin Arg Ile Gly ttg gea gga atg ogt cca att atg ott Leu Ala Gly Met Arg Pro Ile Met Leu 245 gag Glu 250 ttg ggo ggt aag Leu Gly Giy L~ys gat gca Asp Ala 255 ggt ato gtc tta Gly Ile Val Leu 260 gtt gca ggt got Val Ala Gly Ala 275 got. gat got gao Ala Asp Ala Asp gat aao got got Asp Asn Ala. Ala aaa oaa ato Lys GIn Ile 270 goa att aag Ala Ile Lys 816 864 tat gat tac Tyr Asp Tyr tot S er 280 gga caa cgo tgt Gly Gin Arg Cys acg Thr 285 ogt gtg Arg Val 290 ott gte gtt gaa Leu Val Val Giu gaa Giu 295 gtt gow gat gaa Vai Xaa Asp Giu ttg Leu 300 goa gaa aaa ata Ala Giu Lys Ile ict Ser 305 gaa aai gta gea Giu Asfl Val Ala tta tea gta ggt Leu Ser Val Gly cca tit gat aat Pro Phe Asp Asn gca Al a 320 960 acg gig aca ceg Thr Val Thr Pro gtt Val 325 att gat gat aat Ile Asp Asp Asn tea Ser 330 get gac ttt at Ala Asp Phe Ile gaa age Giu Ser 335 1008 tia gta gia Leu Val Val gat Asp 340 gea cgt caa aaa Ala Arg Gin Lys ggi Gi y 345 geg aaa gaa tig Ala Lys Giu Leu aai. gaa tt Asn Giu Phe 350 cat gtt act His Val Thr 1056 aaa. cgt Lys Arg tta gat Leu Asp 370 ggt cgt cia tta act Gly Arg Leu Leu Thr 360 eca, gga ttg iii Pro Gly Leu Phe gat Asp 365 aig aaa. cia get Met Lys Leu Ala gaa gag cci ttt Giu Glu Pro Phe gga. Gly 380 cca att etc eca Pro Ile Leu Pro 1104 1152 1200 1248 att. Ile 385 att. egt gtc aag Ile Arg Val Lys gat Asp 390 gca gaa gaa. get Ala Glu Glu Ala gtt Val 395 get atti gee aac Ala Ile Ala Asn aaa. Lys 400 tet gat ttt gga Ser Asp Phe Gly ta Leu 405 caa. tea tea gte GIn Ser Ser Val ttt Phe 410 aca. egi gat tie Thr Arg Asp Phe eaa. aaa. Gin Lys 415 gca ttt gat Ala Phe Asp ata Ile 420 gca aat aaa ctt Ala Asn Lys Leu gaa Glu 425 gtt ggt aca gt Val Gly Thr Val cac ait aac His Ile Asn 430 gga etc aaa. Gly Leu Lys 1296 aat aag Asn Lys gga. tet Giy Ser 450 gga egt ggi cew Gly Arg Giy Xaa aat tic eca tie Asn Phe Pro Phe ta Leu 445 1344 1392 ggi gca ggt gt Gly Ala Gly Vai caa Gin 455 ggi ate aga tat Giy Ile Arg Tyr tea. Ser 460 att. gaa gea atg Ile Giu Ala Met a ca Thr 465 aat gia. aaa. teg Asn Vai Lys Ser git etc gat aig Vai Leu Asp Met aaa tag Lys 475 1428 <210> 12 <211> 475 <212> PRT <213> group B streptococcus <400> 12 Leu Thr Lys Glu 1 Tyr 5 Gin Asn Tyr Val Asn Gly Glu Trp Lys 10 Ser Ser Val Asn Gin Phe Val Pro Ile Glu Ile Leu Ser Pro 25 Ile Asp Asp Ser Ser Leu Gly Met Lys Ala Ala Met Thr Arg Glu 40 Glu Val Asp His Ala Gly Arg Glu Ala Leu Pro Trp Ala Ala Leu Thr Val Tyr Glu Arg Ala Gln Tyr Leu His Lys 70 Ala Ala Asp Ile Glu Arg Asp Lys Glu Glu Ile Ala Thr Val Leu Ala Lys Glu Ile 90 Ser Lys Ala Tyr Asn Ala Ser Val Thr Glu Glu Gly 115 Glu 100 Val Val Arg Thr Ala 105 Asp Leu Ile Arg Tyr Ala Ala 110 Gly Lys Met Ile Arg Leu Ser Thr 120 Ser Ala Asp Glu Gly 125 Asp Ala 130 Ser Thr Gly His Lys 135 Leu Ala Val Ile Arg 140 Arg Gin Pro Val Gly 145 Gly Ile Val Leu Ala Ser Lys Ile Ala 165 Ile 150 Ala Pro Tyr Asn Tyr 155 Pro Val Asn Leu Ser 160 Pro Ala Leu Ile Gly 170 Gly Asn Val Val Met Phe 175 Lys Pro Pro Phe Ala Glu 195 Thr 180 Gin Gly Ser Val Ser 185 Gly Leu Val Leu Ala Gly Leu Pro Ala 200 Gly Val Phe Asn Thr 205 Ala Lys Ala 190 Ile Thr Gly Glu Val Asn Arg Gly 210 Ser Glu Ile Gly Asp 215 Tyr Ile Val Glu His Glu 220 Phe 225 lie Asn Phe Thr Gly 230 Ser Thr Pro Val Gly 235 Gin Arg Ile Gly Lys 240 Leu Ala Gly Met Pro Ile Met Leu Glu 250 Leu Gly Gly Lys Asp Ala 255 Gly Ile Val Val Ala Gly 275 Leu 260 Ala Asp Ala Asp Asp Asn Al1a Ala Lys Gin Ile 270 Ala Ile Lys Al1a Tyr Asp Tyr Ser 280 Gly Gin Arg Cys Th r 285 Arg Val 290 Leu Val Vai Giu Giu 295 Val Xaa Asp Giu Leu 300 Ala Giu Lys Ile Ser 305 Thr Glu Asn Val Ala Val Thr Pro Val 325 Leu Ser Val Gly Pro Phe Asp Asn Ala 320 Ile Asp Asp Asn Ser 330 Ala Asp Phe Ile Giu Ser 335 Leu Val Val Lys Arg Asp 355 Asp 340 Ala Arg Gin Lys Ala Lys Glu Leu Asn Giu Phe 350 His Val Thr Gly Arg Leu Leu Thr 36D Pro Gly Leu Phe Asp 365 Leu Asp 370 Met Lys Leu Ala Trp 375 Glu Glu Pro Phe Gly Pro Ile Leu Pro 380 Ala Ile Ala Asn Lys Ile Arg Val Lys Asp 390 Ala Giu Glu Ala Ser Asp Phe Gly Gin Ser Ser Val Phe 410 Thr Arg Asp Phe Gin Lys 415 Ala Phe Asp Asn Lys Thr 435 Ile 420 Ala Asn Lys Leu Giu 425 Val Gly Thr Val His Ile Asn 430 Gly Leu Lys Gly Arg Gly Xaa Asn Phe Pro Phe Leu 445 Gly Ser 450 Gly Ala Gly Vai Gin 455 Gly Ile Arg Tyr Ser 460 Ile Giu Ala Met Thr 465 Asn Vai Lys Ser Ile 470 Val Leu Asp Met