CN101613400B - Surface cytoderm protein of streptococcus suis 2-type and preparation method and application thereof - Google Patents

Abstract

The invention discloses a surface cytoderm protein of streptococcus suis 2-type. The number of the surface cytoderm protein in a genebank is gi (146320114). The invention also discloses a preparation method and application of the protein. The protein can be used as a diagnostic target; and a specific antibody of the protein can be detected in a patient infected with the streptococcus suis. Simultaneously, the protein is also an important protective antigen and can be used for preparing vaccine.

Description

A kind of streptococcus suis 2-type superficial cell wall-held protein, Preparation Method And The Use

Technical field

The present invention relates to a kind of bacterioprotein, relate in particular to the superficial cell wall-held protein of a kind of swine streptococcus, also relate to this proteinic preparation method and purposes.

Background technology

Swine streptococcus (Streptococcus suis) belongs to the R crowd of Lan Shi classification, and 35 serotypes are arranged, and is popular the widest with streptococcus suis 2-type, pathogenic the strongest.After Denmark reported people's infection with streptococcus suis the earliest in nineteen sixty-eight, since the seventies in 20th century, the swine streptococcus case was all reported in American-European part country and area, Hong Kong, was main with meningitis mainly, and toxic shock syndrome (TSS) is rare, does not see outbreak of epidemic.China breaks out the people in 1998 and 2005 twice in Jiangsu and Sichuan and infects the streptococcus suis 2-type epidemic situation, and being different from state's stranger's cases of infection is main characteristics with the meningitis, and clinical manifestation is shock type and meningitis type.Most cases are died from toxic shock syndrome (TSS), and it is impaired that pathological change mainly shows as the many organs of whole body, septicemia companion disseminated intravascular coagulation (DIC).People's pig streptococcicosis has become a kind of new zoonosis of serious threat China people ' s health, and how effectively preventing and control streptococcus suis infection has become the research difficult problem that scientists faces.

Owing to understand very few to virulence factor and protective antigen; The bacterial strain of same serotype also is not quite similar in different regional virulence; Highly pathogenic streptococcus suis infection lacks the effective diagnosis target: such Pathogen Isolation is identified main rely on traditional separation and Culture and biochemical identification, carry out the agglutination test somatotype with the swine streptococcus hyper-immune serum then.But domestic animal is extensively carried suis, and many bacterial strain virulence are low, and it is far from being enough therefore only leaning on biochemical reaction to detect swine streptococcus, must set up the detection means to special virulence factor, to distinguish low virulent strain and virulent strain.Above situation has all hindered development and the timely diagnosis and the control to infecting of effective swine streptococcus disease vaccine.

Make a general survey of the development status of external swine streptococcus disease vaccine, mainly experienced full bacterial immunity and antigen protein two stages of immunity.The nineties in last century; There is the investigator to carry out intravenous injection and can stimulates antibody [the Holt ME that is produced conditioningization by immune swine with the swine streptococcus of Superlysoform deactivation; Enright MR, AlexanderTJ. (1990) Immunisation of pigs with killed cultures of Streptococcus suis type 2. Res Vet Sci.48:23-7].The investigator is also arranged through screening avirulent swine streptococcus two mutants; Wait the whole-bacterial-vaccine [KebedeM that obtains to have protectiveness like temperature-sensitive mutation body, streptomycin dependence two mutants; Chengappa MM; Stuart JG. (1990) Isolation and characterization oftemperature-sensitive mutants of Streptococcus suis:efficacy trial of the mutantvaccine in mice.Vet Microbiol.22:249-57.Foster N; Staats JJ; Chengappa MM. (1994) Isolation; Characterization and protection studies in mice of astreptomycin-dependent mutant of Streptococcus suis type 1/2.Vet Res Commun.18:155-63.], but the full bacterial immunity regular meeting of deactivation causes spinoffs such as immune syndrome.The follow-up investigator of having utilizes swine streptococcus to be correlated with virulence factor muramidase-released protein (MRP), extracellular factor (EF) combination water-in-oil emulsion (WO) and aluminum hydroxide adjuvant (AH) as vaccine; Its protectiveness and toxic side effect have been done evaluation; Find to have equal effectively anti--MRP and resist-the EF titre with the pig of MRP+EF/WO immunity and pig with full bacterium/WO immunity; And in the survival pig after immunity; Do not observe tangible clinical sign [Wisselink HJ; Vecht U; Stockhofe-Zurwieden N, Smith HE. (2001) Protection of pigs against challenge with virulent Streptococcus suis serotype 2strains by a muramidase-released protein and extracellular factor vaccine.Vet Rec.148:473-7.].The hemolysin (SLY) that the human purifying is also arranged is as vaccine immune mouse; Discovery can produce complete provide protection to mouse, avoids injury [Jacobs AA, the Loeffen PL of virulent strain; Van den BergAJ; Storm PK. (1994) Identification, purification, and characterization of athiol-activated hemolysin (suilysin) of Streptococcus suis.Infect Immun.62:1742-8.].But the virulence factor of swine streptococcus more often between since just demonstrate the relative diversity on the space-time; Though MRP, EF and virulence height correlation; But different with European pathogenic strain is that most Canadian pathogenic strains are not expressed MRP and EF, and the 2-type swine streptococcus deletion mutant of MRP and EF is the same with wild-type possesses virulence [Gottshalk M, Lebrun A equally; Wisselink HJ; Dubreuil JD, SmithHE, Vecht U. (1998) Production of virulence-related proteins by Canadian strainsofStreptococcus suis capsular type 2.Can J Vet Res 62:75-79.].China's epidemic strain and non-epidemic strain are all expressed MRP; And experimentation on animals shows that epidemic strain cultivation excretory supernatant does not cause pathology; The virulence factors such as MRP, EF, SLY of depending merely on existing understanding are not enough to explain the pathogenesis of China 2-type swine streptococcus, use MRP to produce in the antibody as people's immune vaccine and possibly be difficult to reach the ideal effect with MRP.And at home; The development of swine streptococcus vaccine also is in the starting state; Be deactivation whole-bacterial-vaccine [Wang Zhuo, Shu Xiuwei, Wang Wencheng. the optimization and the safety testing thereof of pig streptococcicosis bivalent inactivated vaccine candidate strain culture condition. the beastly magazine .38:34-36. of traditional Chinese medicines in (2004)].Therefore be necessary the practical situation according to China, development is fit to the new generation vaccine of the 2-type swine streptococcus of China, and excavating the new immunogenic virulence factor that has is the important channel of seeking new generation vaccine.

In general, surface exposure albumen and cell walls attachment protein often are used as the candidate's target and the serodiagnosis reagent of vaccine.Over nearly 10 years; Also there is the investigator to attempt to seek the candidate molecules of immunogenic swine streptococcus superficial cell wall-held protein as vaccine; Separation such as Haataja S in 1996 also identify a kind of semi-lactosi inhibition type adhesin, in the swine streptococcus of 23 kinds of serotypes, all can detect the existence of this adhesin with the method for immunoblotting; Discovery has very strong immunogenicity and conditioning functions; Be suitable as vaccine candidate molecule [Haataja S, Tikkanen K, Hytonen J; Finne J. (1996) The Gal alpha1-4Gal-binding adhesin of Streptococcus suis, a gram-positivemeningitis-associated bacterium.Adv Exp Med Biol.408:25-34.].People such as Okwumabua O screened through the full genome to 2-type swine streptococcus in 2005; Find that a kind of molecular weight is a 38kDa albumen; Have good immunogenicity and protectiveness; Think that this albumen is vaccine candidate molecule and be suitable as the exploitation of diagnostic reagent preferably; This proteic biological function and with the relation of pathogenesis further [Okwumabua O, Chinnapapakkagari S. (2005) Identification of the gene encoding a 38-kilodalton immunogenic and protectiveantigen of Streptococcus suis.Clin Diagn Lab Immunol.12:484-90] in the research.

Obtaining to have aspect the immunogenic albumen at present; Also lack strong means, the traditional biological method identifies that immunogenic protein is difficult, needs to make up gene library; Use corresponding serum screening then; This method is consuming time longer, can not realize high flux screening, and possibly have false positive reaction.The immunoprotein group is a kind of method of fast, efficiently screening the candidate diagnosis target; Advantage through the 2D electrophoretic resolution is high and the direct identification of protein of mass spectrum combines; Directly choose immunoreactive protein site is arranged on the 2D running gel; Identify with mass spectrometry method, therefore, by the immunoprotein group section of learning to do then fully possible within a short period of time of screening and evaluation immunogenic protein rapidly and efficiently; Especially concerning swine streptococcus is studied less relatively pathogenic micro-organism, more likely identify a plurality of brand-new immunogenic proteins in the short period of time.

Summary of the invention

The invention discloses a kind of streptococcus suis 2-type novel surface cell wall protein, Preparation Method And The Use.The disclosed streptococcus suis 2-type superficial cell of the present invention wall-held protein has the aminoacid sequence shown in the sequence 1 in the sequence table, and the gene of this superficial cell wall-held protein of encoding has the nucleotide sequence shown in the sequence 2 in the sequence table.This albumen called after SSU98_0267, its genebank is numbered gi|46320114.This proteic gene of encoding extensively exists in the strong virulence bacterial strain of streptococcus suis 2-type.

This superficial cell wall-held protein is the new immunogenicity antigen molecule of identifying through the immunoprotein group of 2 type swine streptococcus; Present result according to the swine streptococcus genome annotation; The prediction of this gene open reading frame is only arranged; The report that this albumen of Shang Weiyou is expressed in swine streptococcus does not have the clue of this gene function yet.

The invention also discloses the recombinant expression plasmid of expressing above-mentioned streptococcus suis 2-type superficial cell wall-held protein, it contains the nucleotide sequence of sequence 2 in the ordered list.Above-mentioned expression plasmid can be colibacillus expression plasmid, and concrete plasmid can be pET32a etc.

The invention also discloses the bacterial strain of the recombinant expression plasmid that contains above-mentioned expression streptococcus suis 2-type superficial cell wall-held protein, this bacterial strain is intestinal bacteria, can be the BL21 bacterial strain.

The invention also discloses the preparation method of above-mentioned streptococcus suis 2-type superficial cell wall-held protein, this method comprises the steps:

At first clone this superficial cell wall-held protein gene: can carry out through PCR method, at first design synthetic primer, can add restriction enzyme site and protectiveness base at the primer two ends; Then according to PCR method; Template with the swine streptococcus preparation increases under certain condition, and amplified production is carried out agarose gel electrophoresis; And reclaim product and carry out enzyme and cut, confirm superficial cell wall-held protein gene.Be the recombinant plasmid preparation of expressing this superficial cell wall-held protein gene then: the superficial cell wall-held protein gene of preparation is connected with coli expression carrier; Expression vector can be prokaryotic expression carrier, can adopt commercial prokaryotic expression carrier, like coli expression carrier pET32a; The preparation expression vector; And transformed into escherichia coli, preparation reorganization bacterium utilizes bacterium colony PCR and restriction enzyme recombinate screening and the evaluation of bacterium; The reorganization bacterium is carried out the IPTG abduction delivering; Ultrasonic disruption reorganization bacterium is collected supernatant, carries out purifying with GE nickel affinity chromatography post.

The invention also discloses the purposes of above-mentioned superficial cell wall-held protein.This albumen can be used as the diagnosis target, and in animal and human's serum of streptococcus suis infection, obviously rise, thereby can be used for developing the diagnostic reagent of streptococcus suis infection to this proteic antibody, be again important protective antigen simultaneously, can be used for preparing vaccine.The invention also discloses the application of superficial cell wall-held protein in preparation streptococcus suis 2-type vaccine.The whole blood killing experiments of the immune serum conditioning of this protein Preparation finds that this albumen has suitable protectiveness, can be used as emphasis vaccine candidate and diagnosis molecule.

The present invention at first is accredited as the new immunogenicity antigen molecule of streptococcus suis 2-type with this superficial cell wall-held protein.Prepared this antigen and prepared corresponding antibodies through gene engineering method.Whole blood BA through the antibody conditioning confirms: the immune serum of this protein Preparation can significantly improve the germicidal action of people's whole blood; Suitable with the conditioning germicidal action of the full bacterial immunity serum of swine streptococcus; Be new efficient protective antigen, can be used as emphasis vaccine candidate molecule.

Description of drawings

Fig. 1 is reorganization plasmid enzyme restriction collection of illustrative plates.Wherein 1 is dna molecular amount standard DL15000, and 2 is that recombinant plasmid is through EcoRI and XhoI enzyme double digestion.

Fig. 2 is the expression of recombinant plasmid collection of illustrative plates, and wherein 1-5 is for after inducing, and 6 for before inducing, and 7 is the LMWP standard, is followed successively by 97.4kDa from top to bottom, 66.2kDa, 43.0kDa, 31.0kDa, 20.0kDa.

Fig. 3 is to the comparison of SSU98_0267 specific antibody.The antibody horizontal of patient and healthy subjects is (A) relatively, and the antibody horizontal before and after pig infects is (B) relatively.The ELISA experiment is used to estimate the special antibody response to SSU98_0267, and Trx (Trx) is as negative control.

Embodiment

Embodiment one streptococcus suis 2-type surface detail cell wall

The expression and purification of Protein S SU98_0267 and Antibody Preparation

1 material and method

1.1 bacterial strain and plasmid

Streptococcus suis 2-type 98HAH12 strain [Chen C; Tang J; Dong W; Wang C, Feng Y, et al (2007) A Glimpse of Streptococcal Toxic Shock Syndrome from ComparativeGenomics of S.suis 2Chinese Isolates.PLoS ONE 2 (3): e315]; BL21 is the international standard strain; Expression vector pET32a (+) is the Novagen Company products.

1.2 enzyme and reagent

PCR agents useful for same such as restriction enzyme such as EcoRI and XhoI and Taq enzyme, dNTPs, DL15000 molecular weight standard are precious biotechnology (Dalian) ltd product; It is that the sky is Company products that DNA reclaims test kit; The nickel affinity chromatography post is the GE Company products; It is the V-gene Company products that plasmid extracts box; Freund's complete adjuvant and Freund's incomplete adjuvant are the Sigma Company products; Amp is middle promise medicine company product.

1.3PCR design of primers and SSU98_0267 nucleotide sequence, protein sequence

According to the fragment that will increase, design a pair of primer, the primer two ends are added EcoRI and XhoI restriction enzyme site and protectiveness base respectively, primer P1 and P2 streptococcus suis 2-type SSU98_0267 gene ORFs (ORF) the total length segment that can increase.Primer sequence is respectively:

Upstream primer P1:5 '-tagaattcagcaagcagaaagttgtgtc-3 ' sees sequence 3 in the sequence table;

Downstream primer P2:5 '-gcctcgagttcttcttttttgtttttgaatag-3 ' sees sequence 4 in the sequence table.

Nucleotide sequence＞SSU98_0267 sees sequence 2 in the sequence table.

Protein sequence＞SSU98_0267 sees sequence 1 in the sequence table.

1.4PCR amplification

Add the go forward side by side performing PCR amplification of each reactant successively by following order and condition.Streptococcus suis 2-type template DNA 2 μ L, 10 * buffer, 5 μ L, 2.5mmol/L dNTPs 3 μ L, each 1 μ L of primer P1 and P2 (5 μ mol/L), ddH ₂O 37 μ L, LA-Taq enzyme (5U/ μ L) 1 μ L.The condition of amplification does; 94 ℃ of 7min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 7min.

1.5PCR the recovery and the enzyme of product are cut

The PCR product is at 1% agarose gel electrophoresis, and the back is reclaimed test kit with DNA and reclaimed the purpose fragment, and cuts 3h with EcoRI and 37 ℃ of enzymes of XhoI.

1.6 construction of recombinant plasmid and evaluation

The single bacterium colony of DH5 α intestinal bacteria of pET32a empty plasmid is carried in inoculation in the LB meat soup that contains 100 μ g/mL Amp; After 37 ℃ of shakes are cultivated 12～16h; 1% switching is gone into 37 ℃ of shakes of fresh LB meat soup of 100 μ g/mL Amp and is cultivated 6h, with plasmid extraction test kit extracting plasmid.Behind EcoRI and the XhoI double digestion, reclaim test kit recovery enzyme with DNA and cut product.The PCR double digestion is reclaimed product be connected with empty plasmid double digestion recovery product, and the DH5 α host bacterium of transformed competence colibacillus.The preparation of competence bacterium, transform and all to be undertaken, utilize bacterium colony PCR and digestion with restriction enzyme recombinate screening and the evaluation of bacterium by ordinary method.

1.7 the order-checking of recombinant plasmid and sequential analysis

The sequencing of recombinant plasmid adopts the terminal cessation method of the two deoxidations of Sanger, and three rich polygala root Bioisystech Co., Ltd accomplish by Beijing, verifying its reading frame, and carries out homology analysis with BLAST software.

1.8 expression and the purifying of recombinant plasmid in intestinal bacteria

Recombinant plasmid by the e. coli bl21 of ordinary method transformed competence colibacillus, is utilized Amp resistance screening reorganization bacterium, choose in single colony inoculation LB meat soup and (contain 100 μ g/mL Amp), 30 ℃ of violent shakes are cultivated 2～4h, as bacterial concentration OD ₆₀₀Reach at 0.5～0.6 o'clock, add IPTG to final concentration 1mmol/L, 30 ℃ are continued violent shake and cultivate 2～4h.The centrifugal 10min of 6000r/min precipitates resuspendedly with zero(ppm) water, adds isopyknic 2 * electrophoresis sample-loading buffer and boils 10min, and SDS-PAGE detects.Also equally after IPTG induces processing, SDS-PAGE detects expression to the e. coli bl21 that contains sky pET32a (+).With ultrasonic disruption inductive reorganization bacterium, collect supernatant, carry out affinity chromatography with GE nickel affinity chromatography post, concrete steps are undertaken by working instructions.

1.9 recombinant protein animal immune test

The albumen of collecting is diluted to 0.1mg/ml with the PBS of PH7.210mmol/L, adds the Freund's complete adjuvant of equivalent, subcutaneous immune Wistar rat (available from Military Medical Science Institute experimental animal center), 0.5ml/ is only; Later on 3-5 week, respectively with 0.2,0.4,0.8,1.6mg/ml adds the Freund's incomplete adjuvant booster immunization, and the docking of the 7th week is got blood ELISA method and surveyed antibody titer.Set up full bacterial immunity control group and blank group (the pET32a empty plasmid transforms BL21, and abduction delivering Trx albumen is blank with this protein immunization rat) simultaneously.Prepare antiserum(antisera) with ammonium sulfate precipitation method.

2. result

2.1PCR result: the PCR product band of a 2298bp occurs behind electrophoresis, size is consistent with expection.

2.2 the evaluation of recombinant plasmid

The SSU98_0267 gene fragment is through reclaiming, with EcoR I and Xho I restriction enzyme site directed cloning to pET-32a, acquisition recombinant plasmid, called after SSU98_0267-pET-32a.The DH5 α host bacterium of recombinant plasmid transformed competence colibacillus obtains the bacterium of recombinating through the Amp resistance screening.Extract recombinant plasmid,, can cut out the segment about about 3000bp, explain that the SSU98_0267 fragment successfully clones, see Fig. 1 through EcoR I and Xho I double digestion.

2.3 the expression of recombinant plasmid

Reorganization BL21 transformed bacteria is after IPTG induces, and thalline SDS-PAGE shows the protein band of a 100kD, and contain bacterium before inducing at this place no specific band, see Fig. 2.Obtain the SSU98_0267 albumen of purifying through ni-sepharose purification.

2.4 sequencing result and sequential analysis

Clone's sequence length for 2298bp, shown in sequence in the sequence table 2, is 765 amino-acid residues after the translation through order-checking, sees sequence 1 in the sequence table.

Embodiment two streptococcus suis 2-type superficial cell wall-held proteins

The immunogenicity of SSU98_0267 and antibody-mediated opsono-cytophagic test

1.1 reorganization streptococcus suis 2-type superficial cell wall-held protein SSU98_0267 and many anti-preparations: see embodiment one.

1.2SSU98_0267 antibody-mediated opsonophagocytosis

Blood agar is scraped and is got streptococcus suis 2-type list bacterium colony, inserts the THB substratum, and 37 ℃, CO ₂The fresh THB culture medium culturing 8h of switching was to logarithmic phase after incubator was cultivated 8h.Get 1ml bacterium liquid (about 1 * 10 ⁸CFU/ml) 13,000rpm, 1min collection bacterium, PBS washes bacterium and resuspended, is adjusted to 10 ⁶CFU/ml is coated with blood agar, is designated as initial bacterium amount.(≈ 10 to get 50 μ l ⁶CFU/ml) bacterium liquid adds 100 μ l antibody, 15min on ice behind 37 ℃ of 15min; Add 37 ℃ of 350 μ l healthy subjects whole bloods, 3h; Add 55 μ l 1%saponin, 20min on ice; The dilution of piping and druming back is coated with blood agar repeatedly, blood agar numeration mono-clonal behind the 16h.

1.3 infected pigs and philtrum SSU98_0267 albumen specific antibody are measured

Adopt in EUSA (ELISA) mensuration patient and the infection animal and be directed against this proteic specific antibody, adopt the serum of healthy subjects and infect preceding animal serum as contrast.Adopt the recombinant protein of 5 μ g/ml to encapsulate elisa plate, encapsulating damping fluid is 0.05M NaHCO ₃(PH 9.6), 4 ℃ encapsulate and spend the night, and adopt 37 ℃ of sealings of 3%BSA 3h then.The dilution in 1: 500 of patient and healthy subjects serum sample, porcine blood serum dilution in equal 1: 200 before and after infecting, reaction signal is through two anti-detections of horseradish peroxidase (HRP) mark.

2. result

2.1 the conditioning phagocytosis that recombinant protein is antibody-mediated

Obtain purified recombinant Protein S SU98_0267 through GE nickel affinity chromatography column chromatography, add respectively Fu Shi fully with the Freund immunity after, prepare antiserum(antisera), carry out indirect BA.Sterilizing rate calculates: (CFUrTrx-CFUr98_0267)/and CFUrTrx.Trx is the Trx from the e. coli bl21 ni-sepharose purification that contains pET32a (+) empty carrier.

Antibody-mediated opsono-cytophagic test: use the SSU98_0267 recombinant protein respectively; Rat blood serum behind the MRP recombinant protein immune rat is nursed one's health healthy human blood, carries out the fragmentation test of people's whole blood to the streptococcus suis 2-type viable bacteria.The antiserum(antisera) conditioning human whole blood of SSU98_0267 recombinant protein is about 50% to the kill rate of streptococcus suis 2-type; But a little less than the proteic antiserum(antisera) of MRP, the proteic antiserum(antisera) conditioning human of MRP whole blood is about 90% to the kill rate of streptococcus suis 2-type under the similarity condition.

2.2 the SSU98_0267 specific antibody detects in people who infects and the porcine blood serum:

We have detected the situation that exists of SSU98_0267 specific antibody in patient that 6 streptococcus suis 2-types infect and 5 the healthy subjects serum, and the result finds to compare with healthy subjects, and patient SSU98_0267 specific antibody level obviously raises, see Fig. 3 A.We have detected before and after the streptococcus suis 2-type infected pigs situation that exists of SSU98_0267 specific antibody in the serum, the result find with infect before compare, infect that SSU98_0267 specific antibody level significantly raises in the serum of back, see Fig. 3 B.Above-mentioned experimental result shows that SSU98_0267 can be used as the diagnosis target and is used for the serodiagnosis that streptococcus suis 2-type infects.

Pig chain surface cell wall protein 0267.SEQ

Sequence table

< 110>Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL

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gatactccgc aaacaccaca agcgccatca actcctaaag aggaagcacc gaaggaagaa 1560

gttccaacac caccggctcc atcagtacca gaagagcaac caaaagaaac tccaacacca 1620

gaagttccaa aacaagaaga tgttcaaccg gaagcgccaa aatctgataa agtagaatct 1680

gacaaacaaa tgccagaaac taagaaacca gatatgaagc aaccgaaggc agacgacatg 1740

cctaaggaac aaaagccgaa agccgacgag ccaaaggcag agcaaccaca aatggacaaa 1800

ccacaaatgg aagctcctaa gaaagattcg gaagcgccaa aatctgataa agtagaaact 1860

gacaagcaat tgccagaaac taagcaacca gatatgaagc aaccgaaggc agacgacatg 1920

cctaaggaac aaaaaccgaa agccgacgag ccaaaggcag agcaaccaca aatggacaaa 1980

ccacaaatgg aagctcctaa gaaagattcg gaagcaccaa aatctgataa agtagaaact 2040

gacaaaccaa tgccagaaac taaacaacca gatatgaagc aaccgaaggc agataaacca 2100

gaagcagaga aagctcaaat gccacggaca gagggtatga agcctgaatc taaggcttca 2160

atgatgccaa aagcagaggc accaaaagca actttgccaa atacaggcga agcaagtagc 2220

gcaataggct ggctaggtgg tgctttagcc actcttgcca caggcttgta tctattcaaa 2280

aacaaaaaag aagaatag 2298

<210>3

<211>28

<212>DNA

<213>

<400>3

tagaattcag caagcagaaa gttgtgtc 28

<210>4

<211>32

<212>DNA

<213>

<400>4

gcctcgagtt cttctttttt gtttttgaat ag 32

Claims (10)

1. streptococcus suis 2-type superficial cell wall-held protein, it is characterized in that: its aminoacid sequence is shown in sequence in the sequence table 1.

2. according to the superficial cell wall-held protein of claim 1, it is characterized in that: the nucleotide sequence of its encoding sox is shown in sequence in the sequence table 2.

3. recombinant expression plasmid of expressing the said streptococcus suis 2-type superficial cell of claim 1 wall-held protein is characterized in that containing the nucleotide sequence of sequence 2 in the ordered list.

4. recombinant expression plasmid according to claim 3, wherein expression plasmid is colibacillus expression plasmid pET32a.

5. a bacterial strain of expressing the said streptococcus suis 2-type superficial cell of claim 1 wall-held protein is characterized in that containing the said recombinant expression plasmid of claim 3.

6. according to the said bacterial strain of claim 5, wherein expression strain is an e. coli bl21.

7. the preparation method of claim 1 or 2 said superficial cell wall-held proteins comprises the steps:

(1) clones this superficial cell wall-held protein gene;

(2) gene is connected transformed into escherichia coli with colibacillus expression plasmid;

(3) abduction delivering;

(4) expression product is carried out separation and purification.

8. according to the said method of claim 7, wherein colibacillus expression plasmid is PET32a, and intestinal bacteria are E.coli BL21.

9. claim 1 or the 2 described superficial cell wall-held proteins application in preparation streptococcus suis 2-type infection diagnostic reagent.

10. claim 1 or the 2 described superficial cell wall-held proteins application in preparation streptococcus suis 2-type vaccine.

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