CN104762246A - Establishment and application of Streptococcus suis type 2 cell wall protein antibody detection method - Google Patents
Establishment and application of Streptococcus suis type 2 cell wall protein antibody detection method Download PDFInfo
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- CN104762246A CN104762246A CN201510093096.5A CN201510093096A CN104762246A CN 104762246 A CN104762246 A CN 104762246A CN 201510093096 A CN201510093096 A CN 201510093096A CN 104762246 A CN104762246 A CN 104762246A
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- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
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- C12Y304/21062—Subtilisin (3.4.21.62)
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Abstract
Belonging to the technical field of veterinary microbiology and animal immunology, the invention discloses cloning of Streptococcus suis type 2 cell wall protein gene main functional domain fragment and application of the recombinant protein expressed by the fragment in Streptococcus suis type 2 antibody detection. The invention prepares an expression strain Escherichia coli BL21/pET28a-sspA able to secrete Streptococcus suis type 2 cell wall protein SspA, and the preservation number is CCTCC NO: M2014344. The invention also discloses establishment of an SspA recombinant protein based ELISA detection method and application in detection of Streptococcus suis type 2 antibodies. A large number of SSPA antibodies exist in swine infected serum, and the antibody of the protein cannot be detected in immune serum. The method provided by the invention can be employed to detect swine serum antibody and can distinguish Streptococcus suis type 2 infection and immune animals.
Description
Technical field
The present invention relates to veterinary microbiology and animal immunology technical field, be specifically related to based on cell wall protein SspA(Subtilisin-like serine protease, SspA, subtilyne sample serine protease) recombinant escherichia coli strain
escherichia colibL21/pET28a-SspA, and the pig 2 type suis recombinant protein utilizing this bacterial strain to express, set up ELISA detection method, be suitable for the detection with pig 2 type streptococcal infection antibody.
Background technology
Swine streptococcus (Streptococcus suis) is a kind of important pathogenic bacteria that can cause people, pig and other zoogenetic infections, morbidity.According to capsular polysaccharide somatotype, swine streptococcus has 35 serotypes, i.e. 1/2 type and 1-34 type.Wherein streptococcus suis 2-type is virulence the widest the strongest, popular serotype.Pig 2 type suis mainly causes the disease such as meningitis, bronchitis, endocarditis, sacroiliitis, abscess of pig, and weanling pig can be caused dead.After people infects suis, meningitis, endocarditis, septicemia can be caused and permanently to become deaf, the death of people time serious, can be caused.After Dutch reported first people in 1968 infects meningitis case, there is people more than 200 because of infected pigs's suis death.China has had 20 Duo Ge provinces and cities to occur or popular since the seventies, this disease extensively exists and severe epidemic in China, become a kind of principal disease of current serious harm pig industry development, not only caused tremendous economic to lose to pig industry, and cause serious harm to the mankind.
At present for the research of the streptococcic mechanism of causing a disease of pig 2 type, mainly concentrate on its virulence factor.The streptococcus suis 2 type Major Virulence Factors identified has capsular polysaccharide (CPS), muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin (SLY) etc.As glutamate dehydrogenase (GDH), 38 kDa albumen, secretion nuclease (SsnA), swine streptococcus cell wall protein (SspA) etc., and some other potential virulence factor is also relevant to the virulence of swine streptococcus.Because the streptococcic mechanism of causing a disease of pig 2 type it be unclear that, still lack effective vaccine and responsive diagnostic method, Streptococcus suis fails to be controlled effectively always.
In order to prevention and corntrol streptococcus suis 2-type is effectively sick, while booster immunization prevention, use diagnostic method that is special, responsive, quick, convenient operation, provide strong means and important instrument for effective prevention and corntrol streptococcus suis 2-type.At present, the streptococcic prevention and control of pig 2 type are still based on inactivated vaccine immunity, and the antibody that this deactivation vaccine produces plays a very good protection to streptococcus suis infection.The evaluation method of swine streptococcus serology antibody is the indirect ELISA of antigen just like agar diffusion test and based on full bacterium, capsular polysaccharide, Hua Zhong Agriculture University have developed the immunoassay kit of pig 2 type streptococcal capsular polysaccharide antibody, and apply for and obtain patent CN201210245250.2, can be used for detecting swine streptococcus serum antibody, the method all can detect the antibody that inactivated vaccine and wild virus infection produce, and cannot judge pig 2 type streptococcal infection antibody or immune antibody.
In general, Surface exposed proteins and cell wall attached proteins are often by as the candidate targets of vaccine and serotype diagnostic reagent.SspA albumen is arrived by the technical evaluation of Immune inducing in vivo antigen, is a kind of cell wall protein, great expression in infection body, and can not react with immune serum, thus may as a diagnostic marker.In view of these features of SspA albumen, the present invention adopts clone
sspAthe functional domain fragment that gene is important, great expression is purified into SspA albumen, this albumen is utilized to develop quick, accurate, safe, cheap discriminating pig 2 type suis immune swine and the diagnostic kit of wild virus infection pig, for the streptococcic diagnosis of pig 2 type and control provide strong means and instrument.
Summary of the invention
First object of the present invention is that acquisition one strain can secrete the recombination bacillus coli of streptococcus suis 2-type cell wall protein, utilizes this recombination bacillus coli to obtain recombinant protein, utilizes this recombinant antigen protein to prepare pig 2 type streptococcus serum antibody detection method.
The technical scheme realizing the object of the invention is, applicant by preparation, obtain a strain can secrete streptococcus suis type 2 immune protective antigen recombination bacillus coli (
escherichia coli) BL21/pET28a-SspA, this bacterial strain has been delivered on July 21st, 2014 and has been deposited in China typical culture collection center (CCTCC) in the Wuhan University of Wuhan City, Hubei Province, and deposit number is CCTCC NO:M2014344.
Described antigen subtilyne sample serine protease SspA is expressed by the nucleotide sequence genes encoding shown in sequence table SEQ ID NO:1 and obtains.
For making technical solution of the present invention openly complete, recombination bacillus coli of the present invention (
escherichia coli) building process of BL21/pET-28a-SspA is as follows:
(1) extraction of pig 2 type suis genomic dna
By the pig 2 type streptococcus inoculation of this Laboratories Accession on the TSA substratum containing 10% deactivation new-born calf serum, the single colony inoculation of picking, in TSB liquid nutrient medium, is placed in shaking table 150 r/min, 37 DEG C of concussion overnight incubation.Get 1.0 ml bacterium liquid in the centrifuge tube of 1.5 ml, the specification sheets with reference to the bacterial genomes DNA extraction kit of TIANGEN company extracts streptococcus DNA.
(2) amplification of goal gene
According to GenBank having delivered streptococcus suis 2 type bacterial strain
sspAthe sequence (GenBank accession number: CP000407.1) of gene, application Primer Premier 5. 0 software design fragment is
sspAthe primer of (328-3228 bp), dashed part is the restriction enzyme site introduced
ecor I He
sali.
Primer is as follows:
SspA upstream primer U 5' – GC
gAATTCtggcattggtatatgcgcttccgat – 3'
Downstream primer D 5' – C
gTCGACcctacggtcaacttagaattgaagC – 3'
Cut protection base containing enzyme outside the primer restriction enzyme site of upstream and downstream, be respectively GC and C base.Primer is synthesized by Shanghai Sheng Gong biotechnology company.The DNA of extraction is carried out pcr amplification by the system of 50 μ l.PCR reaction product is detected with 1.2% agarose gel electrophoresis.The results are shown in Figure 3, amplified band size is consistent with expection amplified fragments size.
(3) structure of recombinant expression and screening
By the pcr amplification product of goal gene fragment through corresponding endonuclease digestion, and by pET28a carrier through same double digestion, carry out recovery purifying respectively, transformation of E. coli DH5 α after T4DNA ligase enzyme connects.With bacterium liquid screening positive clone, the plasmid of positive colony is checked order.Be transformed into by recombinant plasmid thermal shock in e. coli bl21 competence, final acquisition carries the prokaryotic expression carrier of object fragment.To pET28a-
sspAclone products is used
ecor I He
sali carry out double digestion qualification with expect size consistent, show further goal gene successful clone in expression vector.
(4) abduction delivering, the purifying of pig 2 type suis SspA albumen
Transformed bacteria is inoculated in the LB agar plate containing 50 μ g/ mL kantlex (Kan), 37 DEG C of overnight incubation.Be seeded to 20 mL containing in the LB substratum of 50 μ g/ mL kantlex by cultivating bacterium liquid 1:100,37 DEG C, 220r/min is cultured to OD
600when value reaches 0.5-0.7, in nutrient solution, add inductor IPTG be 0.5 mmol/L to final concentration and establish negative control.28 DEG C, 180r/min cultivates 6h.Collect the bacterium liquid after induction, after the centrifugal precipitation thalline multigelation obtained 3 times, thalline is resuspended in 5 mL bacteria lysis damping fluids, and adds N,O-Diacetylmuramidase, place on ice, with ultrasonication 5 min.By the thalline centrifugal collecting precipitation after fragmentation, i.e. thick inclusion body, with 10 mmol/L Tris-HCl washing precipitations, dispel with rifle head, vortex washing makes precipitation fully dissolve, centrifugal collecting precipitation, repeated washing 2 times, the recombinant protein of preliminary purification is added 0.3% N-sarcosyl (SKL) to dissolve, abundant vibration, 4 DEG C of dissolvings are centrifugal after spending the night, get supernatant, dialyse with phosphate buffered saline buffer under 4 DEG C of conditions, period changes liquid at least 3 times, collected by centrifugation supernatant is in dialysis tubing, concentrate with protein concentration centrifuge tube, filtration sterilization obtains the albumen of purifying, packing,-20 DEG C save backup.SDS-PAGE electrophoretic analysis shows, under the induction of IPTG, transforms containing recombinant plasmid
e.colibL21 have expressed the fusion rotein of about 112 kDa, and expression product size is consistent with expection.
(5) western blot analyzes
Albumen, after the SDS-PAGE electrophoresis of 10%, is transferred on NC film with semi-dry transfer system by expression product, closes 2h, then carry out Western Blot analysis with the TBS shaken at room temperature containing 1% BSA.By pig 2 type suis rehabilitation serum, hyper-immune serum, health pig serum (being so kind as to give by Microbiological Lab of country of Hua Zhong Agriculture University) and the serum that adsorbs completely with 1% bovine serum albumin (BSA) by suitable dilution proportion.Add the serum of 1:1000 dilution in 4 DEG C of overnight incubation, the anti-shaken at room temperature 1h of the goat-anti pig two that the HRP diluted with 1:5000 marks, carries out luminous color reaction with ECL.Result shows, the recombinant protein of expression can with infection sero-reaction, there is special band, and can not with immune serum reaction, the expression success of pig 2 type suis SspA albumen.
The invention also discloses recombination bacillus coli described in more than one
escherichia colithe pig 2 type streptococcal antigen subtilyne sample serine protease SspA of BL21/pET28a-SspA secretion is building the application in the ELISA method detecting pig 2 type suis antibody.
The invention also discloses the recombination bacillus coli can secreting pig 2 type streptococcal antigen subtilyne sample serine protease SspA described in more than one
escherichia colibL21/pET28a-SspA application in detection streptococcus suis 2-type subtilyne sample serine stretch protein enzyme antibody.
The object of the invention is to be to provide a kind of ELISA method that can detect pig 2 type streptococcus serum antibody, the feature of the method have employed pig 2 type suis SspA albumen as antigen, described SspA albumen can detect antibody in streptococcus suis infection serum, but can't detect antibody in pig 2 type suis immune serum, test kit is had and can differentiate to distinguish the characteristic infected with immune animal.
For making technical solution of the present invention openly complete, the ELISA method of the detection pig 2 type suis antibody that the present invention builds is:
The foundation of pig 2 type streptococcal cell wall Protein S spA protein ELISA detection method, it comprises the following steps:
Pig 2 type streptococcal cell wall Protein S spA protein ELISA detection method, the antigen that described method is is recombinant protein SspA, and this SspA proteantigen is by design
sspAgene primer carries out pcr amplification to pig 2 type suis genome, and the gene fragment clone obtained pcr amplification, to expression vector, be converted into intestinal bacteria, then abduction delivering purifying obtained;
The foundation of pig 2 type streptococcal cell wall Protein S spA protein ELISA detection kit, it comprises the following steps:
1) skim-milk is dissolved in diluent A, makes the final concentration of its skim-milk be 5%, as the diluent of serum to be checked and control serum.
2) antigen coated microplate (being coated with the check-out console of streptococcus suis 2-type cell wall protein SspA antigen) is got, first wash plate 2 times with the washings diluted, then the serum to be checked, negative control sera and the positive control serum that have diluted respectively are got 100 μ L join in antigen coated plate hole.1 hole established by serum to be checked, and negative control and positive control respectively establish 2 holes.Shake sample in even hole (not overflowing) gently, is placed in 37 DEG C of incubations 30 minutes.
3) discard the solution in hole, every hole adds the washings 200 μ l diluted, and leaves standstill after 3 minutes, discards the washing lotion in hole, and pat dry on thieving paper, repeat to wash plate 5 times, pat dry for the last time on thieving paper.
4) every hole adds goat-anti pig ELIAS secondary antibody 100 μ l, puts 37 DEG C of incubations (same step 3) of method of washing plate after 30 minutes 5 times.
5) every hole adds each one of nitrite ion A, nitrite ion B (about 50 μ l), and mixing, room temperature (18-25 DEG C, identical below) lucifuge develops the color 10 minutes.Every hole adds stop buffer one (about 50 μ l), measures every hole OD in 10 minutes by microplate reader
620read value.
Test kit criterion of the present invention is: in microplate reader, survey each hole OD
620nm value, by calculating sample S/P value judgement yin and yang attribute, being judged to the positive as during sample S/P>=0.249, being judged to feminine gender during S/P≤0.184, S/P be in be judged between the two suspicious.Wherein S/P=(sample OD
620-standard female serum OD
620)/(standard positive serum OD
620-standard female serum OD
620).
Concrete principle of the present invention is: SspA albumen is the albumen utilizing Immune inducing in vivo antigen technology screening early stage, by design
sspA geneinterior segments
sspAthe primer of (328-3228 bp), clonal expression SspA albumen, analyzed by western-blotting, the antibody that target protein obtains in the infected serum of recombinant protein of expression in e. coli bl21 identified, there is good immunocompetence, but the antibody not containing this recombinant protein in immune serum, has pointed out to possess and has distinguished streptococcus suis infection and immune potentiality.By utilizing the SspA albumen of expressing and obtaining, setting up indirect ELISA method and confirming SspA albumen energy as the streptococcic antigen of diagnosis pig 2 type.Secondly, provide a kind of application of enzyme linked immunological kit in the enzyme linked immunological detecting streptococcus suis 2-type SspA albumen of streptococcus suis 2-type SspA albumen, using extract streptococcus suis 2-type SspA albumen as antigen coated on polystyrene micropore plate, then sera incubation to be checked is added, add goat-anti pig enzyme labelled antibody again, two kinds of situations should be there are during colour developing, if serum to be checked is positive, the goat-anti pig enzyme labelled antibody so now added will continue and the antibody response in positive serum, and microplate reader detects numerical value will be higher.If seronegativity to be checked, so goat-anti pig enzyme labelled antibody would not with streptococcus suis 2-type SspA albumen test on elisa plate, the numerical value obtained will be low.This test kit specificity is good, susceptibility is high.Domestic also without similar business-like product at present, there is good application prospect.
The invention has the beneficial effects as follows:
It is provided by the invention that to detect pig 2 type streptococcic detection method to instrument requirements based on SspA proteolytic enzyme be microplate reader, testing process 3 hours consuming time, the method has good Sensitivity and Specificity, the antibody of the streptococcus suis 2-type SspA albumen in the detection porcine blood serum of the large flux of energy, detect and do not need to rely on quantitative real time PCR Instrument, imager etc., detect antigen used to be obtained by genetic engineering bacterium mass propgation, testing cost is low.
Accompanying drawing explanation
Fig. 1: the accession number of the antigen gene sequences that the present invention uses and position residing in suis 05ZYH33 thereof (be shown as in bracket by underscore for described
sspAgene order in the accession number of Genbank, the numeral after colon thereafter for the position of gene order shown in this accession number in suis 05ZYH33 genome, the overstriking italics shown behind this position be described streptococcic bacterial strain number).
Fig. 2: be pET-28a (+) Vector map that the present invention uses.
The pcr amplification schematic diagram of Fig. 3: sspA gene, wherein, M is Marker2000, and 1 is blank, and 2 is SspA (328-3228bp) gene.
The double digestion qualification schematic diagram of Fig. 4: recombinant plasmid pET28a/SspA, wherein, M is Marker15, and 000,1 is blank, and 2 is the qualification of SspA-pET28a double digestion.
The SDS-PAGE of Fig. 5: SspA albumen analyzes schematic diagram, and wherein: M is Protein Marker, 1 does not induce for negative control and empty carrier, and 2 is the product after induction 3h, and 3 is the product after induction 4h, and 4 is the recombinant protein of purifying.
The Western-blotting schematic diagram of Fig. 6: SspA recombinant protein.
Fig. 7: positive serum sensitivity test result schematic diagram.
Embodiment
The present inventor through extensive and deep research, from
s.suis2amplification coding gene in the genome of Sichuan strain isolated SC19
sspAgene fragment, obtains the higher SspA albumen of purity by escherichia coli prokaryotic expression system.In order to make the present invention easier to understand, set forth embodiments of the invention further below in conjunction with subordinate list.The present invention will be further described in conjunction with the embodiments and demonstration.But the present embodiment is not limitation of the present invention.
material below used by the present embodiment:
1) bacterial strain and plasmid:
This tests Ziyang strain isolated when streptococcus suis 2 type bacterial strain used is Sichuan outburst Streptococcus suis in 2005.Recombinant organism strain DH5 α and BL21 is by this testing laboratory conservation.Carrier is that this laboratory of pET28a (+) is preserved, and its collection of illustrative plates as shown in Figure 2.
2) main agents and damping fluid:
Various restriction enzyme and T4 DNA ligase enzyme, PCR related reagent, DNA Marker and glue reclaim test kit, restriction enzyme
ecor I,
sali all purchased from being TaKaRa Products; The little extraction reagent kit of plasmid, N,O-Diacetylmuramidase, IPTG, albumen Marker are purchased from Shanghai bio-engineering corporation; Protein concentration centrifuge tube is purchased from Millipore company; Bacterial genomes DNA extraction kit is purchased from TIANGEN company; Tris-HCl, 0.3% N-sarcosyl (SKL), NC film, 1% bovine serum albumin, GST, kantlex, ECL nitrite ion, SDS-PAGE joins glue test kit purchased from Beijing Suo Lai Science and Technology Ltd.; Two of HRP mark resists, substrate solution A, substrate solution B, and nitrite ion etc. is purchased from Wuhan Ke Qian animal biological product company limited.
Bacteria lysis buffer formulation: Tris 1.51g, EDTA 0.47g, NaCl 1.46g are dissolved in water, adjust PH to 8.0, are settled to 200mL.
Phosphate buffered saline buffer: dipotassium hydrogen phosphate 5.59g and potassium primary phosphate 0.41g, is dissolved in water into 1L, tune pH value is 7.8-8.0.
embodiment 1: the extraction of pig 2 type suis genomic dna
By the pig 2 type streptococcus inoculation of this Laboratories Accession on the TSA substratum containing 10% deactivation new-born calf serum, the single colony inoculation of picking, in TSB liquid nutrient medium, is placed in shaking table 150 r/min, 37 DEG C of concussion overnight incubation.Get 1.0 ml bacterium liquid in the centrifuge tube of 1.5 ml, the specification sheets with reference to the bacterial genomes DNA extraction kit of TIANGEN company extracts streptococcus DNA.
embodiment 2: the amplification of goal gene
According to GenBank having delivered streptococcus suis 2 type bacterial strain
sspAthe sequence (GenBank accession number: CP000407.1, specifically as shown in Figure 1) of gene, application Primer Premier 5. 0 software design fragment is
sspAthe primer of (328-3228 bp), dashed part is the restriction enzyme site introduced
ecor I He
sali.
Primer is as follows:
SspA upstream primer U 5' – GC
gAATTCtggcattggtatatgcgcttccgat – 3'
Downstream primer D 5' – C
gTCGACcctacggtcaacttagaattgaagC – 3'
Cut protection base containing enzyme outside the primer restriction enzyme site of upstream and downstream, be respectively GC and C base.Primer is synthesized by Shanghai Sheng Gong biotechnology company.The DNA of extraction is carried out pcr amplification by the system of 50 μ l.PCR reaction product is detected with 1.2% agarose gel electrophoresis.The results are shown in Figure 3, amplified band size is consistent with expection amplified fragments size.
embodiment 3: the structure of recombinant expression and screening
By the pcr amplification product of goal gene fragment through corresponding endonuclease digestion, and by pET28a carrier through same double digestion, carry out recovery purifying respectively, transformation of E. coli DH5 α after T4DNA ligase enzyme connects.With bacterium liquid screening positive clone, the plasmid of positive colony is checked order.Be transformed into by recombinant plasmid thermal shock in e. coli bl21 competence, final acquisition carries the prokaryotic expression carrier of object fragment.To pET28a-
sspAclone products is used
ecor I He
sali carry out double digestion qualification with expect size consistent, show further goal gene successful clone in expression vector.Fig. 4 is shown in plasmid double digestion qualification after object fragment clone.
embodiment 4: abduction delivering, the purifying of pig 2 type suis SspA albumen
Transformed bacteria is inoculated in the LB agar plate containing 50 μ g/ mL kantlex (Kan), 37 DEG C of overnight incubation.Be seeded to 20 mL containing in the LB substratum of 50 μ g/ mL kantlex by cultivating bacterium liquid 1:100,37 DEG C, 220 r/min are cultured to OD
600when value reaches 0.5-0.7, in nutrient solution, add inductor IPTG be 0.5 mmol/L to final concentration and establish negative control.28 DEG C, 180r/min cultivates 6h.Collect the bacterium liquid after induction, after the centrifugal precipitation thalline multigelation obtained 3 times, thalline is resuspended in 5mL bacteria lysis damping fluid, and adds N,O-Diacetylmuramidase, place on ice, with ultrasonication 5 min.By the thalline centrifugal collecting precipitation after fragmentation, i.e. thick inclusion body, with 10 mmol/L Tris-HCl washing precipitations, dispel with rifle head, vortex washing makes precipitation fully dissolve, centrifugal collecting precipitation, repeated washing 2 times, the recombinant protein of preliminary purification is added 0.3% N-sarcosyl (SKL) to dissolve, abundant vibration, 4 DEG C of dissolvings are centrifugal after spending the night, get supernatant, dialyse with phosphate buffered saline buffer under 4 DEG C of conditions, period changes liquid at least 3 times, collected by centrifugation supernatant is in dialysis tubing, concentrate with protein concentration centrifuge tube, filtration sterilization obtains the albumen of purifying, packing,-20 DEG C save backup.SDS-PAGE electrophoretic analysis shows, under the induction of IPTG, transforms containing recombinant plasmid
e.colibL21 have expressed the fusion rotein of about 112 kDa, and expression product size is consistent with expection, and the result is shown in Fig. 5.
embodiment 5:western blot analyzes
Albumen, after the SDS-PAGE electrophoresis of 10%, is transferred on NC film with semi-dry transfer system by expression product, closes 2h, then carry out Western Blot analysis with the TBS shaken at room temperature containing 1% BSA.By pig 2 type suis rehabilitation serum, hyper-immune serum, health pig serum (being so kind as to give by Microbiological Lab of country of Hua Zhong Agriculture University) and the serum that adsorbs completely with 1% bovine serum albumin (BSA) by suitable dilution proportion.Add the serum of 1:1000 dilution in 4 DEG C of overnight incubation, the anti-shaken at room temperature 1h of the goat-anti pig two that the HRP diluted with 1:5000 marks, carries out luminous color reaction with ECL.Result shows, the recombinant protein of expression can with infection sero-reaction, there is special band, and can not with immune serum reaction, the result is as shown in Figure 6.
embodiment 6: the preparation of antigen coated microplate
The carbonate buffer solution of the restructuring SspA albumen of purifying with pH 9.6 is diluted, be added in polystyrene micropore plate by 100 μ L/ holes, wrap spent the night (14-18 hour) at putting 4 DEG C, next day takes out, and discards the antigen coated liquid in hole, every hole adds 100 μ L confining liquids and closes, act on 2 hours under putting room temperature, discard in hole after confining liquid, dry under putting room temperature, with tinfoil paper sealed membrane, antigen coated microplate is sealed, put-20 DEG C of preservations.
embodiment 7: upright titration determines that best bag is by concentration and best serum dilution
Antigen coated: by SspA protein solution coating buffer 1:1000,1:2000,1:4000-32000 dilute, horizontal coated elisa plate, 100 μ L/ holes, and each concentration bag is by 1 row.
Close: after 96 orifice plates spent the night 4 DEG C take out, the liquid PBST got rid of in hole washs three times, adds the PBST confining liquid containing 5% skimming milk, 100 μ L/ holes, 37 DEG C of effect 2 h.
Add serum sample to be checked: serum is started longitudinally dilution composition square formation with 1:10,1:20,1:40,1:80,1:160,1:320,1:640,1:1280,100 μ L/ holes, 37 DEG C act on 30 minutes.
Add ELIAS secondary antibody: taken out from incubator by enzyme plate, get rid of the liquid in hole, every hole adds 200 μ L PBST washingss, leaves standstill and outwells after 3 minutes, and on thieving paper, pat dry 4 times altogether.Every hole adds goat-anti pig ELIAS secondary antibody 100 μ L, and 37 DEG C act on 30 minutes.
Add substrate solution: get rid of liquid in hole, washing methods, with (4), amounts to washing 5 times.Each reacting hole first adds substrate solution A mono-(50 μ L), adds substrate solution B mono-(50 μ L) again, and mixing, room temperature (18 DEG C-25 DEG C) lucifuge develops the color 10 minutes.
Add stop buffer: each reacting hole adds stop buffer one (50 μ l) after colour developing completes, and reads OD in 10 minutes
620value.
According to OD
620value, maximum according to positive value (P)/feminine gender value (N), the principle that negative background is minimum, determines antigen coated concentration and serum optimum diluting multiple.Result (table 1.1, table 1.2) shows, and the best in quality concentration of envelope antigen is 1.0 μ g/mL, and serum diluting multiple is 1:160.
Table 1.1 positive serum
Table 1.2 negative serum
embodiment 8: defining of the positive and negative value of indirect ELISA
Under optimum reaction condition, detect with 90 parts of negative serums, read OD
620value.Calculate negative sample OD
620the mean value X of value and standard deviation SD, according to principle of statistics, during the S/P value>=X+3SD of sample, is the positive; During S/P value≤X+2SD, it is feminine gender; Fall between then for suspicious.S/P=(sample OD
620-standard female serum OD
620)/(standard positive serum OD
620-standard female serum OD
620).Wherein surveyed standard positive serum OD
620value is 1.197, standard female serum OD
620be 0.0545.Result is as shown in table 2.1, table 2.2.
The ELISA detected result of table 2.1 90 parts of negative serums
The ELISA detected result of table 2.2 90 parts of negative serums
As shown in table 2.1,2.2, its mean value X is 0.054, standard deviation SD is 0.065, is namely judged to the positive during sample S/P >=X+3SD=0.054+3 × 0.065=0.249, be judged to feminine gender during S/P≤X+2SD=0.054+2 × 0.065=0.184, S/P be in be judged between the two suspicious.As surveyed standard positive serum OD
620value is 1.197, standard female serum OD
620yin and yang attribute judging criterion corresponding when being 0.0545 is: the OD of sample
620value is positive when being more than or equal to 0.3389, is negative, falls between as suspicious when being less than 0.2647.
embodiment 9: specific test
Pig Related Bacteria and virus causing disease (PRV, PRRV, HCV, pig 7 type suis, C group's Malian drainage) positive serum streptococcus suis 2-type SspA antibody assay kit are detected, detected result is all negative (table 3).
Table 3 pig Related Bacteria and the sero-fast detected result of virus causing disease
embodiment 10: sensitivity test
Detect streptococcus suis infection rehabilitation positive serum, according to the above-mentioned criterion determined, ELISA detects OD
620value>=0.34 is positive, when extension rate to detected result during 1:640 is still positive (as shown in Figure 7), proves that the method set up has good susceptibility.
embodiment 11: comparative is tested
Clinical serum sample is detected with the enzyme plate of SspA albumen bag quilt, the best bag determined according to embodiment 7 is by concentration and best serum dilution, be standard according to the yin and yang attribute threshold value that embodiment 8 is determined, detect 56 parts and infect serum and 30 parts of immune serums, and compare with commercial streptococcus suis 2-type capsular polysaccharide ELISA antibody assay kit, respectively randomly draw the result display of the comparison of wherein 7 parts in table 4.1, table 4.2.
Table 4.1 SspA albumen bag is detected the comparative result of infected pigs's serum by with capsular polysaccharide bag
Table 4.2 SspA albumen bag is detected the comparative result of immune swine serum by with capsular polysaccharide bag
Result display is infected in serum, and CPS and SspA can detect higher OD
620value, represents that these two kinds of antigens can detect infection antibody; And in immune serum, CPS antibody OD
620value is higher is shown as the positive, and SspA antibody OD620 value lower be negative, illustrate that SspA albumen is different from capsular polysaccharide antigen, the antibody infected in serum can be detected, and in immune serum, can't detect the antibody of SspA, thus SspA albumen has been pointed out to can be used for distinguishing infection and immune serum, differential diagnosis pig 2 type streptococcal infection and immune animal.
Swine streptococcus is a kind of important infecting both domestic animals and human disease pathogen, and a lot of country was all once broken out in the world, wherein pathogenic the strongest with streptococcus suis 2-type again.SspA is a kind of cell wall protein, and in microorganism pathogenic bacteria widely, cell wall protein all plays a role at the different times of bacteriological infection.SspA albumen is abundant expression in the process of streptococcal infection host.SspA full length gene 5079bp, to encode 1692 amino acid, its functional domains has subtilisin S8 peptase site (163-738aa), the C section (737-851aa) of S8 PEPD D UF1034 and Asp, His and Ser avtive spot (251-262aa, 311-321aa, 649-659aa).Prokaryotic expression is also purified into full-length gene and there is certain difficulty, and this research successful expression goes out SspA protein fragments (110-1076aa), covers the functional domain that this albumen is main.In embodiment 5 result display SspA albumen only with rehabilitation sero-reaction, pointed out this albumen to possess to differentiate the potentiality of pig 2 type streptococcal infection with immunity.The present invention by setting up indirect ELISA, and applies the method detection rehabilitation serum and immune serum, and result confirms the method further can to whether infected pigs 2 type suis qualitatively judges.Illustrate that the method can infect suis 2 type to pig and carry out differential diagnosis, the present invention has outstanding feature and marked improvement, has good using value and wide market outlook in effective prevention and corntrol streptococcus suis infection.
Sequence table
<110> Guangxi Center for Animal Disease Control & Prevention, Hu Qiaoyun
The foundation of <120> mono-boar 2 type streptococcal cell wall protein antibodies detection method and application thereof
<130> 2014
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2901
<212> DNA
<213> streptococcus suis 2 type bacterial strain (Streptococcus suis)
<400> 1
tggcattggt atatgcgctt ccgatttcag aataggaatg ctaggggttt gatccttatc 60
tgatcgctcc acatgaaggt ctttttcagg aacctgctca gatacttcct tgtcatttaa 120
ccgtacctta tatttcttca caacagcatt tccaaaactg tcactcaaat caagtttgac 180
tgtatccccg tctttcagac tatgtagctc atagcgaaag gctttttcat tatttccata 240
ctgcctccag aagcctcctc gagctacaaa atcttcgttg atcttcaagg accaataaat 300
tccattatcc ttggcgcttc cggtaagtgt caagtgccca tcagcatctg ccaggaggac 360
tcctgcatac tgattttcct cgtcttcatc ttcttcacta tccactacct gtctttccaa 420
tggtagcacc tcattttcca gctgtaaaac agggttttct gtatcaacca acatctgata 480
tttcttcgtg taagtcaact catctgcctc attgtacaca cttatatcca caacatttcc 540
ctcttcaatc agagtaactg gataggtcaa cttcccatcc tcaaaatagc gtttttcacc 600
accattgatg cgcacagaat gagcttgttc ggtttctgct tctatttcaa atgtaaatag 660
ataagtccca tcttcttgct ttttgaagtc gaccaacccg ctatctagct cacgaccaag 720
actttcttta ttgaaacgaa ttgggtcaat cagccagtta ttgacagtaa tcggaacttc 780
tgtttcctcc tcctcatcca gactctctgg acttgtattg atacgaaggt cctttaattt 840
atttcccttc tcatctaccg cagttaacat gatggctcta ggaaaatcag atttaaagaa 900
gtcttgctca aaactgccgt cttcttttac atctatgatt ttccgctctc ccttaccaat 960
gtctagattg gaatagtaaa gtttgatgtc cttaggaaga ctaacattat cactcacttt 1020
ccctttcaat gtaaaacgcc catttttacc aatctggata acctcttcat cctcttcatc 1080
tttttcaata tcaagatcaa tcgtcggctt ttcattatct accaaataat tcaaggtttt 1140
agtgtaaata aggttgccat ccttatcttt caagattaat tcaaaactat gacgtccatc 1200
tgattgctct ggaatctgga gagatagttg accattctca agagagtaat tcacagcctg 1260
agcatccaag ttagcctgta catcggaaac ttgcgctgag acgttcgctt taatttctac 1320
agttttctta ctgcgaattg ccatcaaatc ttccaaattt ttaaactgga ttacaggcgc 1380
catcaaatct ttttcaacag caaccacatt acccgctata tccatcacct caacacggag 1440
atgattttta gcatcgagtg gcagttccac ttctttgaga tggtaatgta gttgacctgc 1500
atcatctaca accttttcaa caagaagatc ttctccattt agattagctc gaacctccca 1560
aactttatta ttatccttgg ctgtgacgac cagttctcta tggctggata gacgattggt 1620
atcaatcgcg acaatctctg gcttttgatt atcaattttg actggcagat aggtatactg 1680
ataggcccca ttttctttat tttttacccg caatctaaag aaatattgtc cttccggtgc 1740
tggtatattt tcatcattgc ttgcatcgta cactttccca tcccaacggt gcaattctat 1800
cggagtacga agcttggaat actccgatag actttcaaaa taatctacat agcgaacacg 1860
agacagcatg gttcctgtat caattcgcct taaaacagga gcatcttctg tagcttcttt 1920
tacaatatcc aaatcatagt tggtaatatc cctcaatagc gcaaatcgaa caaaggcatt 1980
cccaatctga ctgtccgaat gctggttttg aattgcgata ttgtctggat tgagaggcga 2040
ttgattatct tgaatctttt cacgcccaag ttcaatatag cgcccagact tattgtgttt 2100
atagctagat aaaactgagg tcagtttcaa tttagaactg gtttcccaag ccggtgcatc 2160
aacaatcctt tcttttgacc agtcaccaac aaagccaaaa taaggtatgc tgatgtcaga 2220
ctgcccctcc gtcaatgatt tgaagtaaat atacccttct gcaaactggt ctttcgcttc 2280
tcctgcatcc aatttcaggc gaatggttct cttctctttt ggacctagtt gaatggattg 2340
ctcagaaaga tgaatactcg accccttgat ttctgtcgca tgaatctctt taactacttt 2400
tccagaacgt cctattctat caacaggaac atcttgacta gtcaacactt tcccagctga 2460
aatagcaaag cttctttgtt ggtttcccag attttccaag gttacctcaa attctgtctc 2520
acgccctatt tcttttaatt ccacccctcc ttttagacgg tggtgaagaa tcacatctgt 2580
ttcaaaggct ctatcaatct gcaacagacc agcaccttgt tgtctcggag aattttccaa 2640
agcatgccca gatgaatcta gaacatcaac caaaggggta gcagtattca tcaaaatgat 2700
tctcagcaaa tccatcctag tcatgccttc tggtggtgtc atttggcgaa tccgtggcaa 2760
taaaagtgcg ctggcacccg ccacaattgg cgaagccatc gaagtccctg acatggagcc 2820
ataacgattg tcattcaagg ttgcataaac atcctctccc ggcgccacaa tctctggctt 2880
caattctaag ttgaccgtag g 2901
Claims (4)
1. can secrete the recombination bacillus coli of pig 2 type streptococcal antigen subtilyne sample serine protease SspA for one kind
escherichia colibL21/pET28a-SspA, is characterized in that, this bacterium deposit number is CCTCC NO:M2014344.
2. a kind of recombination bacillus coli can secreting pig 2 type streptococcal antigen subtilyne sample serine protease SspA according to claim 1
escherichia colibL21/pET28a-SspA, is characterized in that, described antigen subtilyne sample serine protease SspA is expressed by the nucleotide sequence genes encoding shown in sequence table SEQ ID NO:1 and obtains.
3. recombination bacillus coli described in a claim 1
escherichia colithe pig 2 type streptococcal antigen subtilyne sample serine protease SspA of BL21/pET28a-SspA secretion is building the application in the ELISA method detecting pig 2 type suis antibody.
4. the recombination bacillus coli of pig 2 type streptococcal antigen subtilyne sample serine protease SspA described in a claim 1, can be secreted
escherichia colibL21/pET28a-SspA is detecting the application in streptococcus suis 2-type subtilyne sample serine stretch protein enzyme antibody.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114755406A (en) * | 2022-03-28 | 2022-07-15 | 南京农业大学 | Streptococcus suis 9 type ELISA antibody detection method and kit thereof |
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| US20070009900A1 (en) * | 1997-07-02 | 2007-01-11 | Aventis Pasteur Ltd | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| CN101613400A (en) * | 2008-06-25 | 2009-12-30 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of streptococcus suis 2-type superficial cell wall-held protein, Preparation Method And The Use |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114755406A (en) * | 2022-03-28 | 2022-07-15 | 南京农业大学 | Streptococcus suis 9 type ELISA antibody detection method and kit thereof |
| CN114755406B (en) * | 2022-03-28 | 2023-10-24 | 南京农业大学 | Streptococcus suis type9 ELISA antibody detection method and kit thereof |
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