CN101418275A - Streptococcus suis type 2 immune protective antigen - Google Patents

Abstract

The invention belongs to the technical field of livestock infectious diseases, and in particular relates to a streptococcus suis type-II immune protective antigen, a method for preparing the same and application thereof. A core technology related to the invention is to prepare an expression strain Escherichia coli BL21/pET-28a-0197 which can excrete the streptococcus suis type-II immune protective antigen protein 0197, wherein the strain is preserved in China Center for Type Culture Collection (CCTCC) with the preserving number of CCTCC NO: M208146. The invention also discloses a method for preparing an antigen protein suitable for the streptococcus suis type-II subunit vaccine and application thereof.

Description

A kind of streptococcus suis 2-type immune protective antigen

Technical field

The present invention relates to vaccine production technical field in the livestock contagious disease.Be specifically related to a kind of streptococcus suis 2-type protective antigen preparation.At the anti-system of streptococcus suis 2-type, the present invention has designed clone, expression and functional verification and the application of a kind of new protective antigen protein gene of encoding.The antigen protein of described genetic expression can improve the ability of pig opposing streptococcus suis 2-type.

Background technology

Swine streptococcus (Streptococcus suis, S.suis) be the main pathogen that causes Streptococcus suis, capsular polysaccharide (CPS) according to bacterium surface can be divided into S.suis 35 serotypes, be respectively 1/2 type and 1～34 type, but also the someone proposes 32 types and 34 types should belong to streptococcus orisratti recently, and should not belong to swine streptococcus, wherein streptococcus suis 2-type is that virulence is the strongest, wherein streptococcus suis 2-type (SS2) is pathogenic strong, popular wide, and this type also is the highest serotype of clinical cross frequence.SS2 is a kind of important zoonosis pathogenic agent, pig meningitis (meningitis), endocarditis (endocarditi), septicemia (septicemia), sacroiliitis (arthritis), serositis (polyserositis), pneumonia (pneumonia) etc. be can cause, weanling pig or growing and fattening pigs sudden death often caused; This bacterium of human infection can cause meningitis, causes permanent deafness, septicemia, endocarditis, even dead.Reported first was found pig 2 type suis suspected cases in Guangdong Province in 1991, and season is broken out this disease in continuous 2 years in full summer in some areas, 1998-1999 Jiangsu Province.Existing enough at present epidemiologic datas confirm that SS2 has constituted serious threat to the aquaculture and the people ' s health of China.

Because lack the epidemiologic data of Streptococcus suis, pathogenesis is unclear, there are not effective vaccine and responsive diagnostic method, fail to be effectively controlled at a lot of national Streptococcus suis always.

Although the pathogenesis to streptococcus suis 2-type still is in research and discovery stage at present, influenced carrying out smoothly of streptococcus suis 2-type vaccine research, the research of this vaccine has still obtained certain progress.The streptococcus suis 2-type vaccine of research mainly contains inactivated vaccine, living vaccine, genetically engineered live vector vaccine and subunit seedling at present, but in actual applications weak point is arranged respectively.Inactivated vaccine has immunoprotection preferably to the homologous swine streptococcus, but since the virulence gene table existence of different strains than big-difference, not good for the protection of allos bacterial strain; Living vaccine can make pig be protected, but can not remove the 2 type swine streptococcus of settling down in tonsilla or joint, can not remove the bacterium in the inapparent infection pig tonsil; Live vector vaccine mainly is one of main direction of current and following vaccine development and exploitation, vaccine has the advantage of conventional live vaccine and inactivated vaccine concurrently, but according to FDA (Food and drug administ ration, FDA) regulation, can not there be anti-medicine plasmid in the living vaccine, and in the humans and animals body, can't usually keep the stability of recombinant plasmid with antibiosis; Subunit seedling has immune efficacy height, and the advantage such as the security of inactivated vaccine is good of live vaccine, and still, the antigenic component of subunit seedling is single, and is limited to the provide protection of other serotypes.

Given this; the present invention gets down to the research of the novel immune protective antigen of streptococcus suis 2-type; the searching immune protective effect is good, and the immunogenic protein that extensively exists in other swine streptococcus serotype beyond the streptococcus suis 2-type, and then is combined into effective swine streptococcus vaccine.

Summary of the invention

First purpose of the present invention is to obtain a strain can secrete the proteic recombination bacillus coli of streptococcus suis 2-type immune protective antigen; utilize this recombination bacillus coli to obtain immunogenicity better protection antigen protein, utilize a kind of being applicable to of this protective antigen protein Preparation to prevent or/and the subunit vaccine of control streptococcus suis 2-type.

The 2nd purpose of the present invention is the application of prepared recombination bacillus coli in the preparation streptococcus suis II-type subunit vaccine.

In order to realize purpose of the present invention; the applicant is by preparation; obtain a strain and can secrete the recombination bacillus coli of streptococcus suis 2-type immune protective antigen (the gene accession number is seen accompanying drawing 1) (Escherichia coli) BL21/pET-28a-0197; this bacterial strain is delivered the Chinese typical culture collection center (CCTCC) that is deposited in the Wuhan University of Wuhan City, Hubei Province on October 9th, 2008, and deposit number is CCTCC NO:M208146.

In an embodiment of the present invention, the applicant provides the detailed preparation method of this new streptococcus suis 2-type immune protective antigen 0197, and described that this protective antigen is proteic may Application Prospect.

More detailed technical scheme is as described in " embodiment ".

Description of drawings

Fig. 1: the accession number of the antigen gene sequences that the present invention uses and (what be shown as underscore in the bracket is the accession number of described three antigen gene sequences at Genebank in residing position in suis 98HAH33, thereafter the numeral behind the colon is the position of gene order shown in this accession number in suis 98HAH33 genome, and the overstriking italics that shows behind this position is a described streptococcic bacterial strain number).

Fig. 2: be pET-28a (+) the carrier collection of illustrative plates that the present invention uses;

Fig. 3: be that recombinant antigen protein 0197 gene PCR of the present invention (1) and recombinant plasmid double digestion are identified figure (2);

Fig. 4: be SDS-PAGE and Western Blot figure behind recombinant antigen protein 0197 purifying of the present invention;

Fig. 5: be the very high antibody horizontal of inducing behind recombinant antigen protein 0197 immune mouse of the present invention;

Fig. 6: be the institute's IgG antibody subclass of the inducing mensuration behind recombinant antigen protein 0197 immune mouse of the present invention;

Fig. 7: be to attack poison (5LD behind recombinant antigen protein 0197 immune mouse of the present invention ₅₀) demonstration of protection effect;

Fig. 8: be to attack poison (20LD behind recombinant antigen protein 0197 immune mouse of the present invention ₅₀) demonstration of protection effect.

Embodiment

Embodiment 1: the clone of streptococcus suis 2-type recombinant antigen protein 0197 and expression

One material

1) plasmid and bacterial strain

PET-28a (+) plasmid (available from Noagen company) that the present invention adopts, colon bacillus BL21 (DE3) competence is available from Chinese Wuhan City, Hubei Province life technology company limited.This pET-28a (+) plasmid map as shown in Figure 2.

Bacterial strain uses therefor of the present invention be streptococcus suis 2-type CVCC606 bacterial strain available from BeiJing, China China Veterinery Drug Inspection Office, belong to a commercial strain.

2) the streptococcus suis 2-type antigen gene is described referring to Figure of description 1.

3) main agents and damping fluid (Buffer)

Sodium-chlor, EDTA disodium salt, ethanol, methyl alcohol, Ponceau S, trichoroacetic acid(TCA) (TCA) are Shanghai traditional Chinese medicines company products; Tris alkali (Tris-HCl), dithiothreitol (DTT) (DTT), glycerine, sodium laurylsulfonate (SDS), acrylamide, ammonium persulphate, Tetramethyl Ethylene Diamine (TEMED), yellow soda ash, sodium acetate (giving birth to worker's biotechnology company limited) available from Shanghai.Glycine, Xylene Brilliant Cyanine G R-250 are available from AMRESCO company.Bovine serum albumin (BSA), pancreatin, proteinase inhibitor (PMSF), formaldehyde are Sigma company product.Proteinase K (stock solution concentration is 20mg/ml, uses liquid concentration to be 1mg/ml) is available from Shanghai China Shun biotechnology company limited; Penbritin (Ampcillin), kantlex (Kanamycin), foetal calf serum, inductor IPTG (sec.-propyl-b-d-thiogalactoside) are Invitrogen company product; Suction nozzle and centrifuge tube are AxyGen company products.Endonucleases such as Taq enzyme and 10 * Taq enzyme Buffer, BamH I, Xhol I and relevant Buffer, T4 ligase enzyme and 10 * T4 ligase enzyme Buffer, Rnase, the centrifugal dna gel of UNIQ-10 pillar reclaim test kit, DNA marker (DL-2000, DL-15000) is precious biological (Dalian) company limited product.

The sheep anti-mouse igg of horseradish peroxidase (HRP) mark, goat-anti pig IgG are available from Sigma company.Substrate solution preparation: substrate solution A:0.006%H2O2 damping fluid; Substrate solution B: get Na2HPO412H2O14.2g, citric acid 10.5g is settled to 500mL with distilled water and is made into 0.1 phosphoric acid salt citrate buffer solution (pH5.0), adds benzidine (TMB) then.During use A liquid and B liquid equal-volume are mixed, mix in back 5 minutes and use, now with the current.

The intestinal bacteria substratum: LB liquid medium and solid medium (every liter contains yeast extract 5g, Tryptones 10g, and NaCl 10g transfers pH to 7.5 with 10mol/L NaOH, 121 ℃ of autoclaving 20min, 4 ℃ of preservations are standby.In per 100 milliliters of LB liquid nutrient mediums, add 1.5g agar and be solid LB substratum, 121 ℃ of autoclaving 20min, 4 ℃ of preservations are standby).

Streptococcus cultures TSB, TSA substratum, RPMI-1640 substratum and Freund's complete adjuvant, Freund's incomplete adjuvant are all available from Sigma company.

Three streptococcus suis 2-type antigen proteins of purifying adopt Ni Sepharose 6 Fast Flow purification columns (GEHealthcare company product).

U.S. XOM white oil: available from the capable emerging capable petrochemical industry company limited of Dongguan City (Exxon Mobil soleagent) PBS damping fluid: NaCl 8.0g, KCl 0.2g, KH ₂PO ₄0.24g, Na ₂HPO ₄* 12H ₂O 3.628g is dissolved in the 800ml distilled water, is 7.4 with the hydrochloric acid adjust pH, and distilled water is settled to 1000ml, 121 ℃ of autoclaving 20min, room temperature preservation.The Western-blotting damping fluid:

Electricity changes damping fluid: 39mmol/L glycine, 48mmol/LTris alkali, 0.037%SDS (electrophoresis level), 20% methyl alcohol.

Preparation 1000mL transfering buffering liquid need take by weighing the 2.9g glycine, 5.8g Tris alkali, and 0.37g SDS adds 200mL

Methyl alcohol adds ddH ₂O to total amount be 1000mL.

TBS (pH8.0) damping fluid: 10mmol/L Tris-HCl, 150mmol/LNaCl.

The TBST damping fluid: adding final concentration in above-mentioned TBS is that 0.05%Tween-20 gets final product.

Ponceau S (10 *) stock solution: the 2g Ponceau S, the 30g trichoroacetic acid(TCA), the 30g sulphosalicylic acid adds ddH ₂O to 100mL.

The plasmid extraction related solution:

Solution I: contain 0.05mol/L glucose, 0.025mol/L Tris HCl (pH8.0), 0.01mol/L EDTA, 121 ℃ of rearmounted room temperatures of autoclaving 20min are standby.

Solution II: contain 0.2mol/L NaOH, 1%SDS, now with the current.

Solution III: get 5mol/L sodium acetate 60mL, glacial acetic acid 11.5mL, water 28.5mL transfers pH to 5.0.

2mol/L NaOH:8g NaOH adds 100mL ddH ₂O.

10%SDS:10g SDS adds 80mL ddH ₂Among the O, the heated and stirred dissolving is settled to 100mL.

5mol/L NH ₄Ac:327.7g NH ₄AC adds ddH ₂Dissolve among the O, be settled to 500mL.

10mol/L NH ₄Ac:655.4g NH ₄AC is dissolved in ddH ₂O is settled to 500mL.

13%PEG8000 (1.6mol/LNaCl): 13g PEG8000,9.35g NaCl is dissolved in ddH ₂O decides 100mL, 121 ℃ of autoclaving 20min.

3mol/L NaAc:80mL ddH ₂Add the 40.81g sodium-acetate among the O, be settled to 100mL, 121 ℃ of autoclaving 20min.

Phenol: chloroform: primary isoamyl alcohol (volume ratio is 25:24:1): get saturated phenol 25mL, chloroform 24mL, primary isoamyl alcohol 1mL, fully behind the mixing, cover, in brown bottle, preserve with TE (pH8.0).

Chloroform: primary isoamyl alcohol (volume ratio 24:1): get chloroform 48mL, primary isoamyl alcohol 2mL, behind the abundant mixing, TE (pH8.0)

Cover, preserve in the brown bottle.

TE solution: 1mmol/L EDTA, 10mmol/L Tris-Cl pH value is 8.0.

Design of primers is with synthetic

Use primer-design software Primer 5.0, the primer that has designed the HP0197 gene that is used to increase is right, and it is synthetic that this primer is given birth to worker Bioisystech Co., Ltd by Shanghai.Primer sequence is as follows:

Forward primer is 5-GCTGAATTCACGACAGAGACTTCAACA (EcoR1);

Reverse primer is 5-TAGCTCGAGGCGCTGTTCACCTGT (XhoI).

Experimental animal adopts female BALB/c small white mouse in 7 ages in week, available from Disease Prevention Control Center, Hubei Prov.

2, antigen protein preparation method

The extraction of streptococcus suis 2-type (SS2) genomic dna

1) streptococcus suis 2-type (SS2) bacterium is cultivated

On the TSA substratum that contains 10% deactivation new-born calf serum (vide ut supra), the single colony inoculation of picking places shaking table (150r/min) in the TSB liquid nutrient medium with CVCC606 strain bacterial classification inoculation, 37 ℃ of concussion overnight incubation.

2) extraction of streptococcus suis 2-type (SS2) genomic dna

Get 1.0ml bacterium liquid in the centrifuge tube of 1.5ml, the centrifugal 5min of 8000r/min abandons supernatant.Precipitation is suspended among the 500 μ lTE (PH8.0), adds 10% SDS, and making its final concentration is 1%, adds 3 μ l Proteinase Ks (20mg/ml) then, 37 ℃ of effect 2h.The centrifuging and taking supernatant, with isopyknic phenol/chloroform extracting 1 time, supernatant with the long-pending dehydrated alcohol of diploid at-20 ℃ of precipitation 10min, the centrifugal 20min of 12000r/min, precipitation washes twice with 70% ethanol, room temperature is placed 20min, allows the alcohol volatilization totally.The deionized water dissolving that precipitation is sterilized with 20 μ l ,-20 ℃ of preservations.

3) amplification of goal gene

The genome of the CVCC606 strain bacterium of extracting with aforesaid method is as pcr template.Primer shown in the use table 1 carries out pcr amplification.The PCR reaction system is as follows:

ddH ₂O 37.5μl

10×Taq?Buffer 5.0μl

dNTP(2.5mM?each) 4.0μl

Upstream primer 1.0 μ l

Downstream primer 1.0 μ l

Dna profiling 1 μ l

Taq enzyme (5U/ μ l) 0.5 μ l

Response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of 1min; 55 ℃ of 1min; 72 ℃ of 5min; 72 ℃ of 10min are extended in 30 circulations.

4) the PCR product reclaims:

The UNIQ-10 pillar DNA glue that adopts the sea to give birth to worker's biotechnology company limited reclaims test kit and reclaims dna fragmentation, reclaims the step of the specification sheets of test kit according to the centrifugal dna gel of UNIQ-10 pillar and carries out, and concrete operations are as follows:

1) with 0.8% agarose gel electrophoresis, target DNA fragment and other DNA are separated as far as possible, under long-wave ultra violet lamp,

Be used in the knife blade that burnt on the spirit lamp flame and downcut the agar block that contains target DNA fragment, put into 1.5ml and sterilize centrifugal

In the pipe.

2) add 400 μ l Binding Buffer by every 100mg agarose gel, put 10min in the 50-60 ℃ of water-bath, make sepharose thoroughly melt (when adding thermosol, every 2min mixing once).

3) the UNIQ-10 post is put into collection tube, the sol solution that melts is transferred in the UNIQ-10 post, room temperature leaves standstill 2min, the centrifugal 1min of room temperature 8000rpm.

4) take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, add 500 μ l Wash Solution, the centrifugal 1min of room temperature 8000rpm.

5) repeating step 4, take off the UNIQ-10 post, outwell the waste liquid in the collection tube, and the UNIQ-10 post is put into same collection tube, the centrifugal 15sec of room temperature 12000rpm.

6) the UNIQ-10 post is put into the 1.5ml centrifuge tube of a sterilization, according to PCR product amount relatively what, the film central authorities in the pillar bottom add 10-20 μ l Elution Buffer or ddH ₂O, room temperature or 37 ℃

Place the centrifugal 1min of 2min room temperature 12000rpm, the liquid in the centrifuge tube is the dna fragmentation of recovery, can use immediately or be stored in-20 ℃ standby.

The structure of recombinant expression plasmid

Utilize EcoR1+Xho1I (available from precious biological (Dalian) company limited product) while enzyme to cut the HP0197 gene fragment and the pET-28a (+) of pcr amplification, reclaim pcr amplification segment and expression vector, enzyme is cut back HP0197 gene fragment cut afterwards with enzyme that pET-28a (+) is connected, construct the pET-28a-0197 plasmid.Above-mentioned restriction enzyme site is single restriction enzyme site (on position is between EcoR1 and Xho1I restriction enzyme site) in the used plasmid pET-28a of the present invention (+).

Connect the conversion of product

Get competent cell DH5 α 100 μ l and join in the 1.5mlEP pipe, each the 5-10 μ l of recombinant plasmid pET-28a-0197 after connecting is added and mixing.After putting on ice 30min, 42 ℃ of heat shock 90sec, ice bath 3min-5min.Add 400 μ l LB, make its recovery in 37 ℃ of 200rpm shaking culture 45min.Recombination bacillus coli suspension after the recovery discards 400 μ l supernatants in 4 ℃ of centrifugal 10min of 5000rpm, coats the LB agar plate that contains 25 μ g/ml Kna with the resuspended precipitation of remaining 100 μ l.37 ℃ of propagation 1h turn over flat board again, are inverted 37 ℃ of cultivation 14h-16h and occur to bacterium colony.

The enzyme of recombinant plasmid is cut evaluation

The extraction of plasmid: the use alkaline lysis (with reference to " molecular cloning experiment guide " third edition. Huang Peitang etc. translate, Beijing, Science Press, the method for 2002 editions introductions) carry out, the concrete operations step is as follows:

1) with sterilization toothpick several single bacterium colonies of picking at random on the LB flat board, be inoculated in respectively in the LB liquid nutrient medium of 3ml 25 μ g/ml kantlex, 37 ℃ of shaking culture are spent the night.

2) bacterium liquid is changed in the 1.5ml centrifuge tube, in 4 ℃ of centrifugal 3min of 8000rpm, abandon supernatant, with remaining 1.5ml bacterium liquid repeated centrifugation, the handstand centrifuge tube flows to end liquid on thieving paper again.

3) add the solution I that 100 μ l ice precooling, vortex fully suspends thalline, adds the solution II of the new preparation of 200 μ l again, put upside down centrifuge tube repeatedly for several times, ice bath 5min adds the solution III that 150.0 μ l ice precooling at last, gentleness is put upside down centrifuge tube for several times, ice bath 10min.

4) in 4 ℃ with the centrifugal 10min of 12000rpm, draw supernatant to another 1.5ml centrifuge tube, add isopyknic Virahol, evenly mixed, room temperature leaves standstill 5min.

5) the centrifugal 10min of room temperature 12000rpm abandons supernatant, and precipitation is with after the 75% cold ethanol rinsing, according to conventional vacuum-drying or seasoning method drying.

6) precipitation is dissolved with the TE (pH8.0) that 200 μ l contain 20 μ l Rnase (20 μ g/ml), and 56 ℃ of water-bath 30min or 37 ℃ of water-bath 1h are to remove RNA.

7) add 7.5mol/L NH4Ac 100 μ l, room temperature leaves standstill 5min, again in the centrifugal 5min of room temperature 12000rpm.

8) draw supernatant in the EP pipe of another 1.5ml, add the cold dehydrated alcohol of 2 times of volumes, ice bath is put 10min.

9) in 4 ℃ of centrifugal 10min of 12000rpm, abandon supernatant, precipitation is dissolved in 20 μ lddH with after the 75% cold ethanol rinsing after the vacuum-drying ₂O or TE (pH8.0) put-20 ℃ of refrigerators and preserve standby.

The double digestion of recombinant plasmid is identified: utilize the EcoR1+XholI enzyme to cut the pET-28a-0197 expression plasmid, should occur after enzyme is cut expecting that the external source fragment and the carrier segment of size are correct recombinant plasmid.

The structure of recombinant strains: get competent cell BL-21 (DE3) 100 μ l and join in the 1.5mlEP pipe, each 0.5 μ l of recombinant plasmid pET-28a-0197 is added and mixing.After putting on ice 30min, 42 ℃ of heat shocks 90 seconds, ice bath 3min-5min.It is coated the LB agar plate that contains 25 μ g/ml Kna.Just putting 1h for 37 ℃, again flat board is being turned over, be inverted 37 ℃ and cultivate 14h-16h and occur to bacterium colony.

Expression and the purifying of goal gene in intestinal bacteria

The abduction delivering of goal gene: the expression strain that will contain recombinant antigen protein 0197 is inoculated in the 3mLLB liquid nutrient medium that contains 25 μ g/ml Kna, cultivates in 37 ℃ of shaking tables.From cultured bacterium liquid, get 100 μ L and be inoculated in 10mL and contain in the fresh LB liquid nutrient medium of 25 μ g/mlKna, in 37 ℃ of about 3h of shaking culture, to OD ₆₀₀When reaching 0.6-1.0, adding IPTG is 0.8mmol/L to final concentration, and continuation is collected thalline after cultivating 3h.

The SDS-PAGE electrophoretic analysis of expression product

The preparation of SDS-PAGE electrophoresis sample:

With the recombination bacillus coli after inducing in the centrifugal 15min of 8000r/min.Precipitate resuspendedly, and add N,O-Diacetylmuramidase to final concentration 1mg/ml, ice bath 30min with the 50mM Tris-cl (pH8.0) of 1/10 volume.Carry out under the condition of ice bath ultrasonic broken broken, until bacterium liquid thickness no longer, 10000r/min, centrifugal 30min.Take a morsel respectively last cleer and peaceful precipitation after the cracking adds 2 * protein electrophoresis sample-loading buffer, 125 μ l, DTT 25 μ l and TE liquid 100 μ l, and the concussion mixing, 100 ℃ are boiled 10min, and the centrifugal 5min of 12000r/min carries out the SDS-PAGE electrophoretic analysis.

The preparation of sds page and electrophoresis

12% separation gel preparation: purified water 1.6ml, 30% acrylamide soln 2.0ml, 1.5mol/L Tris-Base solution (pH8.8) 1.3ml, 10%SDS 0.05ml, 10% ammonium persulphate 0.05ml and TEMED 0.003ml.Each composition is added the back mix rapidly, add in the glue plate, add purified water above.5% spacer gel preparation: purified water 2.1ml, 30% acrylamide soln 0.5ml, 1.0mol/L Tris-Base solution (pH6.8) 0.38ml, 10%SDS 0.03ml, 10% ammonium persulphate 0.03ml, TEMED 0.003ml; Each composition is added the back mix rapidly, above the separation gel of adding glue plate, fill the back and insert the application of sample comb.After treating that spacer gel solidifies, take off comb; Gel sets on electrophoresis apparatus, is added the Tris-glycine electrophoretic buffer of q.s, in well, add each sample respectively; Electrophoretic voltage 200V, electric current are in the 20mA-40mA scope, and electrophoresis 1h to tetrabromophenol sulfonphthalein swimming plastic emitting bottom surface, stops electrophoresis.

Polyacrylamide gel dyeing and decolouring

Unload gel, with coomassie brilliant blue R250 staining fluid (45% methyl alcohol, 45% purified water, 10% glacial acetic acid, 0.25% coomassie brilliant blue R250) dyeing 30min, use destainer (45% methyl alcohol again, 45% purified water, 10% glacial acetic acid) 1min that decolours, observations.

The purifying of recombinant protein

With the recombinant antigen protein of collecting express bacterium use Binding buffer (20 mMTris-HCl pH7.9,5mMImidazole, 0.5MNaCl) resuspended, ultrasonic disruption, 4 ℃ 12, the centrifugal 15min of 000g gets sample on the supernatant.Use Binding buffer and Washing buffer (20mMTris-HCl pH7.9 respectively, 15mM Imidazole, 0.5MNaCl) wash post near the OD value reaches baseline, use Elution buffer (20mMTris-HCl pH7.9 instead, 40mM Imidazole, 0.5MNaCl) recombinant antigen protein of elution of bound, collect the crest Partial Protein and be used for further analysis as the recombinant antigen protein of purifying.

The effect of present embodiment is seen shown in Figure 3.Fig. 4 is seen in recombinant antigen protein HP0197 purifying SDS-PAGE electrophoretic analysis.

Embodiment 2: the specificity analysis of recombinant antigen protein

1. use the antigenicity of Western-blot methods analyst recombinant antigen protein

The recombinant antigen protein 0197 of above-mentioned purifying is carried out the SDS-PAGE electrophoresis according to ordinary method.The steps include:

1) shift: cut out 6 Whatman 3M filter paper and 1 nitrocellulose membrane (NC film), the size of filter paper and film will equate fully with the size of gel or be slightly less than gel, marks for one jiao at filter membrane with pencil, guarantees the relative direction of transfer printing caudacoria and gel; Nitrocellulose membrane is soaked 5min in purified water; In another shallow pallet, add a small amount of transfering buffering liquid, 6 Whatman 3M filter paper are soaked in wherein.According to following steps the electrophoretic blotting groove is installed then: keep flat the base (anode) of Graphite Electrodes, put 3 layers of 3M filter paper, nitrocellulose membrane, polyacrylamide gel and 3 layers of 3M filter paper successively.The bubble of thoroughly getting rid of each interlayer; The loam cake of electrophoretic blotting groove is anchored on Graphite Electrodes-transfer film glue complex body; Connect power supply, according to the parameter making current of gel slab area according to 0.65mA/cm2-1.0mA/cm2, electrophoretic transfer 0.5h-2h.

2) ponceau dyeing: shift finish after, take off the NC film, in deionized water, after the rinsing 2 times-3 times, be transferred to the 5min-10min that dyes in the ponceau staining fluid, observe the transfer printing effect, and mark albumen Marker position with pencil; Use the rinsed with deionized water nitrocellulose membrane until color fade under the room temperature;

3) the NC film is placed 5% skim-milk, room temperature sealing 2h;

4) wash film: abandon confining liquid, wash the NC film 3 times, each 5min with 1 * TBST;

5) anti-hatching: the NC film is put into the anti-SS2 type of the rabbit bacterium positive serum (volume ratio 1:50) that dilutes with 5% skim-milk, hatch 2h for 37 ℃;

6) wash film: take out the NC film, wash film 3 times, each 10min with 1 * TBST;

7) two anti-hatching: film changed over to the sheep anti-mouse igg antibody (volume ratio 1:5000) of the HRP mark of 5% skim-milk dilution, hatch 2h for 37 ℃;

8) wash film and take out the NC film, wash film 3 times, each 10min with 1 * TBST;

9) colour developing: the NC film is placed the DAB colour developing liquid of new configuration, put the dark place colour developing, after the color depth for the treatment of protein band reaches requirement, wash with termination reaction with 1 * TBST.

2. the sero-fast preparation of recombinant protein

1) mensuration of recombinant protein concentration

With reference to " molecular cloning experiment guide " third edition, Huang Peitang etc. translate, Beijing, and Science Press, the method for version introduction in 2002 with the dissolving of PBS damping fluid, is by volume carried out doubling dilution with bovine serum albumin then.Get the protein standard liquid 100 μ l of each concentration respectively, join in the Coomassie brilliant blue dye solution of 5ml, behind the reaction 10min, survey absorbancy at wavelength 595nm place, according to the OD value that records and the relation of respective concentration, the drawing standard curve, and derive reduction formula.

2) preparation of vaccine and immunity test

The preparation of recombinant antigen protein vaccine:, make that the antigen protein final concentration is 500 μ g/ml in the vaccine with recombinant antigen protein 0197 and Freund's complete adjuvant equal-volume mixing and emulsifying.Immunity is for the second time adopted Freund's incomplete adjuvant with vaccine.

Immunization method: give described test mice abdominal cavity inoculation Freund's complete adjuvant emulsive recombinant protein vaccine (inoculum size is 100 μ l/); 2 all pneumoretroperitoneum inoculation Freund's incomplete adjuvant emulsive recombinant protein vaccines (inoculum size is 100 μ l/); Serum in the eye socket bloodletting, was collected after 10 days in the interval.

Adopt Western-blot methods analyst recombinant antigen protein in the present embodiment, the invention effect is seen Fig. 4.

Embodiment 3: the test of recombinant antigen protein mouse immune protection

1. Recombinant Protein Expression and purifying

The expression strain that will contain the pET-28a-0197 plasmid is inoculated in the 3mL LB liquid nutrient medium that contains 0.25 μ g/ml kantlex, cultivates in 37 ℃ of shaking tables.From cultured bacterium liquid, get 100 μ L and be inoculated in 10mL and contain in the fresh LB liquid nutrient medium of 2.5 μ g kantlex, in 37 ℃ of about 3h of shaking culture, to OD ₆₀₀When reaching 0.6-1.0, adding IPTG is 0.8mmol/L to final concentration, and continuation is collected thalline after cultivating 3h.

With the recombinant antigen protein of above collection express bacterium with Binding buffer (20mMTris-HCl pH7.9,5mMImidazole, 0.5MNaCl) resuspended, ultrasonic disruption, 4 ℃ 12, the centrifugal 15min of 000g gets sample on the supernatant.Use Binding buffer and Washing buffer (20mMTris-HCl pH7.9 respectively, 15mM Imidazole, 0.5MNaCl) wash post near the OD value reaches baseline, use Elution buffer (20mMTris-HCl pH7.9 instead, 40mM Imidazole, 0.5MNaCl) recombinant protein of elution of bound, collect the recombinant protein of crest Partial Protein as purifying.

2. the mensuration of recombinant protein concentration (Bradford method)

Embodiment 2 is seen in concrete operations.

3. recombinant antigen protein vaccine production and immunization method

1) preparation of recombinant antigen protein vaccine

With recombinant antigen protein 0197 and Freund's complete adjuvant equal-volume mixing and emulsifying, make that the antigen protein final concentration is that 500 μ g/ml are used for first immunisation in the vaccine, Freund's incomplete adjuvant is used in immunity for the second time, and all the other components are exempted from identical with head.

Inactivated vaccine preparation: after pure culture, 8～10 of the single bacterium colonies of picking are applied on the TSA flat board and (contain 10% deactivation bovine serum) with streptococcus suis 2-type CVCC606 strain bacterium.Behind 37 ℃ of cultivation 16h～18h, the lawn on the flat board is washed, be diluted to 7.5 * 10 with the 0.01mol/L PBS that sterilizes ⁹Cfu/ml with formalin-inactivated (3 ‰) 24h～48h, presses the emulsification of 1:1 volume mixture with freund's adjuvant then, and final concentration reaches 3.0 * 10 ⁹Cfu/ml.

2) immunization method

Test mice peritoneal immunity Freund's complete adjuvant emulsive antigen protein 100 μ l/ only; 2 all pneumoretroperitoneum immunity Freund's incomplete adjuvant emulsive antigen protein 100 μ l/ only.

3) medium lethal dose (LD ₅₀) mensuration

(1) trial test: in prerun, try to achieve 8 ages in week female BALB/c mouse complete dead or 90% or more dead dosage and animal is not dead or 10% below the dosage of death, respectively as the highest and lowest dose level of official test.

(2) injection system: abdominal injection.

(3) animal grouping: set up 4 dosage groups, every group of 6 BALB/c mouse.

(4) dosage: will all be scaled denary logarithm by the highest, the lowest dose level that trial test draws, then will be the highest, the logarithmic difference of lowest dose level, by needed group of number, be divided into the dosage group of several logarithms equidistant (or not equidistant).

(5) calculating of test-results and statistics.

4) test grouping and immunity

In 7 ages in week, 30 of the female BALB/c small white mouses of body weight 18 ± 2g are equally divided into 3 groups, vaccine I and organize (0197), freund's adjuvant control group and PBS control group, 20 every group.Abdominal injection, per 2 week immunity 1 time, totally 2 times, immunizing dose be 0.1ml/ only, during weekly docking get blood, be used for the monitoring of serological specificity antibody.

5) serological specificity detection of antibodies

By elisa plate, 1%BSA37 ℃ of sealing 1h, washings are packaged in-20 ℃ of preservations after washing plate 1 time to the recombinant antigen protein 0197 250ng/100 μ l that uses purifying in 4 ℃ of bags that spend the night.With the mouse blood sampling separation of serum of booster immunization after one week, get 100 μ l behind the doubling dilution and add elisa plate, establish freund's adjuvant contrast and blank simultaneously.37 ℃ of reaction 30min.Add volume ratio 1:5 after washing plate 3 times, sheep anti-mouse igg (the H+L)-HRP of 000 dilution, 37 ℃ of reaction 30min.Add 100 μ l substrate solutions after washing plate 5 times, add the 0.25%HF termination reaction behind the lucifuge colour developing 10min, in the 630nm reading.Get the OD value greater than 0.3 serum maximum dilution multiple as serum antibody titer.

6) recombinant antigen protein inducing mouse IgG antibody subclass is measured

With the mouse blood sampling separation of serum of booster immunization after 2 weeks, get 100 μ l behind the doubling dilution and add elisa plate, 37 ℃ of reaction 30min.Each dilution serum adds volume ratio 1:5 respectively after washing plate 3 times, sheep anti-mouse igg 1-HRP, the IgG2a-HRP of 000 dilution, 37 ℃ of reaction 30min.Add 100 μ l substrate solutions after washing plate 5 times, add the 0.25%HF termination reaction behind the lucifuge colour developing 10min, in the 630nm reading.Get the OD value greater than 0.3 serum maximum dilution multiple titre as this IgG subclass.

7) immunized mice is attacked poison back protection situation

The mouse of booster immunization after two weeks carried out challenge test.Challenge test is divided into two batches to carry out, and 10 every group, the mouse of each protein immunization inoculates 5LD respectively ₅₀And 20LD ₅₀The CVCC606 strain bacterium of dosage.Observe a week Clinical symptoms and the death condition of record mouse continuously.

The expression of recombinant antigen protein, purification effect are seen Fig. 4 in the present embodiment; CVCC606 strain bacterium medium lethal dose (LD ₅₀) be 4 * 10 ⁸CFU; Recombinant antigen protein 0197 is carried out the serological specificity detection of antibodies find to induce very high antibody horizontal, effect is seen Fig. 5; Recombinant antigen protein inducing mouse IgG antibody subclass is measured, and the results are shown in Figure 6.With recombinant antigen protein 0197 immune mouse of purifying, with 5 LD ₅₀With 20 LD ₅₀Attack poison and estimate the immune protective efficiency of recombinant antigen protein, the result as shown in Figure 7.。

The present invention is by clonal expression streptococcus suis 2-type antigen protein 0197, and the characteristic to 0197 has proved that through the row experimental analysis this antigen protein has better immunogenicity, can induce higher antibody horizontal behind the immune mouse, and mainly induce the immunity of Th2 type.Attacking malicious protection experiment and show that this albumen can obviously strengthen the resistibility of mouse to SS2, is a kind of good immune protective antigen, can be used as the candidate antigens of streptococcus suis II-type subunit vaccine.

Claims (5)

1, a kind of recombination bacillus coli (Escherichia coli) BL21/pET-28a-0197 that can secrete the streptococcus suis 2-type antigen protein is deposited in Chinese typical culture collection center (CCTCC), and deposit number is CCTCC NO:M208146.

2, a kind of by the described recombination bacillus coli excretory of claim 1 streptococcus suis 2-type antigen protein.

3, a kind of subunit vaccine by the preparation of the described recombination bacillus coli excretory of claim 2 streptococcus suis 2-type antigen protein.

4, the application of the described recombination bacillus coli of claim 1 in preparation streptococcus suis 2-type antigen protein.

5, the application of the described recombination bacillus coli of claim 1 in the preparation streptococcus suis II-type subunit vaccine.

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