CN101979406B - Multi-epitope protein for South Africa type II foot-and-mouth disease, and preparation method and application thereof - Google Patents
Multi-epitope protein for South Africa type II foot-and-mouth disease, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a multi-epitope VP1-VP3 protein for South Africa type II foot-and-mouth disease, a prokaryotic expression plasmid pGEX-VP1-VP3 constructed by a nucleotide sequence encoding the multi-epitope protein and a prokaryotic expression vector pGEX-6P-1, a recombinant expression strain obtained by transferring the pGEX-VP1-VP3 into Escherichia coli BL21(DE3)plysS, and a detection method for performing enzyme cutting and purification on the protein expressed by the recombinant expression strain and establishing a South Africa type II foot-and-mouth disease specific enzyme-linked immunosorbent assay (ELISA) by using the purified expressed protein. The method has the advantages of high specificity, high sensitivity, high speed, simplicity, convenience, reliability and the like, and is suitable for quickly detecting a plurality of samples.
Description
Technical field
The present invention relates to gene engineering technology field, specifically, relate to a kind of South Africa many epitopes of II type foot and mouth disease VP1～VP3 albumen, its preparation method and application.
Background technology
Foot and mouth disease (Foot-and-mouth disease, FMD) is artiodactylous a kind of acute, the height contagious disease that is caused by foot and mouth disease virus (FMDV), is once classified as first of the category-A epidemic disease by OIE (OIE).Foot and mouth disease belongs to the world pop sexually transmitted disease, and its outburst has caused great harm to global human health and livestock industry production.
FMDV is the single strand plus RNA virus of icosahedron, genome is about 8.5kb, contain a large open reading frame, be divided into L, P1, four zones of P2 and P3, the capsid protein VP4 of P1 district coding virus wherein, VP2, VP3 and VP1, the VP4 of each 60 molecule, VP2, VP3 and VP1 consist of viral capsid proteins, the VP1 major part is exposed to the surface of virus particle, it is the main component that determines virus antigenicity, but the inductive infection animal produces neutralizing antibody, it is inner that the VP4 major part is embedded in virus, and VP2 and VP3 are between VP4 and VP1, and four kinds of structural polypeptides all participate in immunogenic formation.VP1～VP3 forms capsomere, and VP4 is positioned at virion inside.The Nonstructural Protein of 3 kinds of viruses of P2 genes encoding, 2A, 2B, 2C.P3 genes encoding nonstructural protein 3A, Vpg, 3Cpro and 3Dpol.
Foot and mouth disease virus has seven serotypes, i.e. O, A, C, SAT I, SAT II, SATIII and Asia I type.These seven serotypes can be divided into two groups according to homology, O, A, C and Asia I type are first group, SAT I, SAT II, SATIII are second group, various homology reaches 60%～70% in the group, but homology only is 25%～40% between two groups, and no cross reaction between each serotype of foot and mouth disease virus is even in same serotype, the antigenicity of different virus also has difference, and this has increased difficulty just for quarantine and the prevention and control of foot and mouth disease.
Traditional foot and mouth disease diagnosis and quarantine method mainly contain complement fixation test (CFT) (CFT), indirect hemagglutination test (PHA), agar diffusion test (AGP), virus neutralization tests (VNT), the serological methods such as enzyme linked immunosorbent assay (ELISA), along with molecular biological development, the application of RT-PCR technology and biochip technology makes the detection of foot and mouth disease virus towards future development more responsive, special, easily and fast.Nineteen fifty-two, Brooksby has set up CFT, and the method that viral Isolation and proliferation and complement fixation test (CFT) combine was continued to use nearly 30 years in the foot and mouth disease diagnosis, although this technology accurately and reliably, but responsive not, and have anticomplementary activity in some sample, impact detects effect.Indirect hemagglutination test is take red corpuscle as carrier, according to the antigen of sensitized erythrocyte, the characteristic of antibody, and available known antigen or antibody or the antigen of antibody test the unknown.At present existing commercial foot and mouth disease indirect hemagglutination diagnostic reagent box is widely used in the antibody test of foot and mouth disease.But agar diffusion test is detectable antigens both, also detectable antibody.According to antigen-antibody phase mutual diffusion in gel, produce principle, available known antigen or the corresponding antibody of antibody test or the antigen of precipitation line in optimal proportions.Virus neutralization tests (VNT) also claim serum neutralization test, and VNT both can identify antigen, but again antagonist quantitative assay has type specificity, is the most classical foot and mouth disease detection method, is the gold standard of estimating other method.The advantages such as the RT-PCR technology is used for the detection of foot and mouth disease virus, has that susceptibility is high, specificity good, quick, and is accurate, the PCR product can also be used to order-checking, and then obtains more detailed epidemiology information, gives information for research hoof-and-mouth disease poison strain develops.
ELISA detects foot and mouth disease and has the advantages such as specificity is good, susceptibility is strong, quick, easy, reliable, and can be automated operation, the rapid detection that is suitable for a large amount of samples is subject to people's attention in the diagnosis of foot and mouth disease day by day, has now become one of ordinary method that detects in the world foot and mouth disease.The method of traditional detection aftosa serum antibody mainly is that the FMDV that adopts bhk cell to cultivate is the indirect ELISA method of antigen, although specificity and the sensitivity of this method are higher, but in the production process of virus, easily cause the diffusion of virus, exist many potential safety hazards, need the BSL-3 laboratory environment.For this reason, many investigators at expression in escherichia coli structural protein and the Nonstructural Protein of FMDV, it as detectable antigens, has been set up the indirect ELISA detection method of multiple FMDV.
Africa is the natural focus of foot and mouth disease, and South Africa II type (SAT II type) foot and mouth disease never strides across the equator propagation in history, and this is so that some scholars think that South Africa type foot and mouth disease needs the special living environment in Africa.But occurred in Kuwait and Saudi South Africa II type foot and mouth disease epidemic situation in 2000, make it is found that, SAT II type foot and mouth disease not only can stride across the equator, also can stride across Red sea, arrives Asia continent.Particularly along with the contact of African expanding economy and world market is day by day frequent, opening gradually that the African continent is external also will make the epidemic disease that is settled in Africa originally to disseminating all over the world.
Along with establishing diplomatic relations and trade contacts frequent of China and African country, China is subjected to the threat of South Africa type foot and mouth disease increasing, and wherein SAT II type is maximum to the threat of China.The at present research aspect foot and mouth disease of China is confined to O, A, C, Asia I type foot and mouth disease virus, and for the research of SAT II type, comprises that detection technique, vaccine are in space state fully.
The present invention is adopting SAT II type FMDV structural protein VP1, VP2, VP3 antigen epitope genes flexible amino acid series connection and is carrying out in intestinal bacteria on the basis of amalgamation and expression, set up the special serotype detection method of SAT II type foot and mouth disease virus with it as envelope antigen, to satisfy specific detection, the monitoring requirements of relevant animal and product.
Summary of the invention
The purpose of this invention is to provide a kind of South Africa many epitopes of II type foot and mouth disease VP1～VP3 albumen.
Another object of the present invention provides the preparation method of these many epitope proteins.
A further object of the present invention provides the application of these many epitope proteins.
In order to realize the object of the invention, the invention provides a kind of South Africa many epitopes of II type foot and mouth disease virus VP1～VP3 albumen, it has aminoacid sequence shown in SEQ ID NO:1 or this sequence through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
The present invention also provides the gene of coding above-mentioned South Africa many epitopes of II type foot and mouth disease virus VP1～VP3 albumen, and its nucleotide sequence is shown in SEQ ID NO:2.
The present invention also provides the expression vector that contains above-mentioned nucleotide sequence.
Aforesaid expression vector, its carrier that sets out is pGEX-6P-1.
The present invention also provides the recombinant expressed bacterium that contains above-mentioned nucleotide sequence.
Aforesaid recombinant expressed bacterium, its starting strain are BL21 (DE3) plysS.
The present invention also provides the preparation method of South Africa many epitopes of II type foot and mouth disease virus VP1～VP3 albumen, it comprises the steps: 1) dna fragmentation shown in the SEQ ID NO:2 is connected among the carrier pMD-19T, behind BamHI and SalI double digestion, be connected structure with the pGEX-6P-1 of the same double digestion of process and obtain expression vector pGEX-VP1～VP3; 2) with step 1) expression vector pGEX-VP1～VP3 be converted into e. coli bl21 (DE3) plysS competent cell, screening positive clone and abduction delivering recombinant protein; 3) enzyme is cut and purification step 2) recombinant protein.
The present invention also provides the application of South Africa many epitopes of II type foot and mouth disease virus VP1～VP3 albumen in detecting South Africa II type foot and mouth disease virus.
The present invention provides a kind of test kit for detection of described many epitopes VP1 of containing of South Africa II type foot and mouth disease virus～VP3 albumen envelope antigen in addition.
The invention has the advantages that, adopt the inventive method to prepare South Africa many epitopes of II type foot and mouth disease virus VP1～VP3 albumen, safety coefficient is higher, and is environmentally friendly, and the preparation method is simple and easy to do; Adopt test kit of the present invention to detect South Africa II type foot and mouth disease virus, have the advantages such as specificity is good, susceptibility is strong, quick, easy, reliable, be suitable for the rapid detection of a large amount of samples.
Description of drawings
Fig. 1 is the structure schema of recombinant expression plasmid pGEX-VP1～VP2 of the present invention;
Fig. 2 is the SDS-PAGE result schematic diagram of recombinant bacterium expression product of the present invention, and wherein, M is protein labeling, and 1 is empty carrier bacterium abduction delivering, and 2 do not induce contrast 3 and 4 for recombinant expressed bacterium is the recombinant expressed bacterium fusion rotein that 3h expresses after inducing;
Fig. 3 is fusion rotein of the present invention carries out Western Blot with anti-GST monoclonal antibody detected result schematic diagram, and wherein, M is protein labeling, and 1 is GST-VP1～VP3 fusion rotein, and 2 is the GST carrier proteins;
Fig. 4 is fusion rotein of the present invention carries out WesternBlot with SAT II type FMDV positive serum detected result schematic diagram, and wherein, M is protein labeling, and 1 is GST-VP1～VP3 fusion rotein, and 2 is the GST carrier proteins;
Fig. 5 is that the enzyme of South Africa many epitopes of II type foot and mouth disease virus VP1 of the present invention～VP3 albumen is cut and the purification result schematic diagram, wherein, M is protein labeling, 1 and 2 is blank, 3 is liquid phase direct enzyme cutting product, 4 and 5 is the pGEX-VP1-VP3 fusion rotein, and 6 is GST albumen, and 7 is purpose antigen behind the purifying.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
The structure of embodiment 1 South Africa many epitopes of II type foot and mouth disease virus prokaryotic expression carrier pGEX-VP1～VP3
Adopt flexible amino acid linker to connect good six peptide section VP2 (60-73), VP2 (163-176), VP3 (58-71), VP3 (127-140), VP1 (132-146), the VP1 (199-211) of antigenicity, the corresponding dna fragmentation of chemosynthesis, its nucleotide sequence is shown in SEQ IDNO:2.Then, above-mentioned dna fragmentation is connected among the cloning vector pMD-19T, with BamH I and Sal I double digestion pMD-19T-VP1～VP3 plasmid, simultaneously the pGEX-6P-1 plasmid is carried out same double digestion, and glue reclaims the purpose fragment of the two, be built into prokaryotic expression carrier pGEX-VP1～VP3 through connection, as shown in Figure 1.
The clone of embodiment 2 South Africa II type foot and mouth disease virus multiple epitopes VP1～VP3 dna sequence dnas
Extract pGEX-VP1～VP3 plasmid, take this plasmid as template, dna sequence dna by pcr amplification South Africa many epitopes of II type foot and mouth disease virus VP1～VP3, classify as according to design primer P1, P2 with the nucleotides sequence of many epitopes, contain respectively BamH I, Sal I restriction enzyme site in P1 and the P2 primer, primer sequence is as follows:
P1：5’-
CGCTTCTTTAAGGA-3’
P2：5’-
CCAGCTGTTTTTCCA-3’
Wherein, BamH I, Sal I restriction enzyme site represent with italic respectively.
The PCR reaction system is: recombinant plasmid 1 μ L, 10 * PCR Buffer (Mg
2+) 2.5 μ L, dNTP 2 μ L, P1, each 1 μ L of P2 primer, rTaq archaeal dna polymerase 0.5 μ L, sterilization ddH
2O is supplemented to 25 μ L.Response procedures: 94 ℃ of 5min; 94 ℃ of 1min, 50 ℃ of 50s, 72 ℃ of 1min carry out 30 circulations altogether; 72 ℃ are extended 10min.
Enzyme is cut evaluation: the little recombinant expression plasmid pGEX-VP1～VP3 that carries, and to carry out enzyme with restriction endonuclease BamH I, Sal I and cut evaluation, the endonuclease reaction system is: plasmid DNA 2 μ g, 10 * T buffer, 7.5 μ L, BamH I 2 μ L, Sal I 2 μ L are with sterilization ddH
2O supplies 50 μ L systems, and 37 ℃ of water-bath enzymes are cut 3h behind the mixing.
Will through PCR identify and enzyme cut evaluation all positive recombinant strains send the center order-checking of Beijing promise match genome research.
The structure of embodiment 3 recombination bacillus coli BL21 (DE3) plysS/pGEX-VP1～VP3
To be converted into e. coli bl21 (DE3) plysS competent cell through the recombinant expression vector pGEX-VP1～VP3 that identifies, coating LB/ penbritin (Amp) flat board, selecting a plurality of bacterium colonies puts into 37 ℃ of LB liquid nutrient mediums and cultivates after 12 hours and use respectively isopropylthio-β-D-galactoside (IPTG) abduction delivering, then carry out SDS-PAGE and Western-blot and detect, result such as Fig. 2 are to shown in Figure 5.Therefrom filter out can be in e. coli bl21 (DE3) recombination bacillus coli BL21 (DE3) plysS/pGEX-VP1～VP3 of abduction delivering South Africa II type foot and mouth disease virus multiple epitopes VP1～VP3 albumen.
Determining of embodiment 4 best inductor concentration and best induction time
The single recombination bacillus coli BL21 of picking (DE3) plysS/pGEX-VP1～VP3 bacterium colony adds penbritin (Amp) to final concentration 50 μ gmL to 3mL LB substratum
-1, after 37 ℃ of shaking tables are cultivated 12h, this bacterium liquid is forwarded to 2 fresh * YT of 3mL by 1% inoculum size (contains 50 μ gmL
-1Amp) in the substratum, 37 ℃ of 200rmin
-1, cultivate OD
600Be about 0.6～0.8, the adding final concentration is 1mmolL
-1IPTG, 37 ℃ of abduction deliverings, and 2h, 4h, 6h, 8h respectively collect 1mL bacterium liquid, 12000rmin after inducing
-1Centrifugal collection thalline.Suspend with 50 μ L pH7.4 PBS, add 2 * sample-loading buffer of equivalent, process 10min, the bacterium specimen chamber temperature 10000rmin after the processing through boiling water bath
-1Centrifugal 3min.Get 10 μ L supernatants and carry out SDS-PAGE, establish and do not induce bacterium liquid in contrast.Electrophoresis is used coomassie brilliant blue staining after finishing, acetic acid decolouring observations.Determine that according to the expression of many epitope proteins best induction time is 6 hours.
After having determined induction time, be respectively 0.1,0.5,1.0 and 1.5mmolL in IPTG concentration
-1Condition under induce 6h, each collects 1mL bacterium liquid, centrifugal collection thalline.Carry out SDS-PAGE after the bacterium sample is processed as stated above and determine the best induced concentration of protein expression.Determine that according to the expression of many epitope proteins the best induced concentration of IPTG is 1.0mmolL
-1
A large amount of expression and purities of embodiment 5 South Africa many epitopes of II type foot and mouth disease virus VP1～VP3 albumen
The single recombination bacillus coli BL21 of picking (DE3) plysS/pGEX-VP1～VP3 bacterium colony is to 3mL LB substratum, adding penbritin to final concentration is 50 μ g/mL, 37 ℃ of shaking table incubated overnight, get the 10 μ L bacterium liquid that spends the night next day and join in the LB substratum that 30mL contains 50 μ g/mL penbritins, put 37 ℃ of shaking tables and be cultured to OD
600Be about 0.6～0.8, adding inductor IPTG is 1mmol/L to final concentration, continuation inducing culture 6 hours.
(pGEX-VP1～VP3) the centrifugal 2min of 30mL 12000r/min collects thalline, adds 3mL MagneGST bacteriolyze reagent, resuspended bacterial precipitation with the bacterium liquid of abduction delivering.The RNase-Free DNase I that adds 300 μ L, room temperature, incubation bacterial suspension 30min on shaking table makes its abundant cracking.With lysate and glutathione agarose
TM-4B (2ml homogenate) filler mixing, under room temperature, shaking table absorption 30min.With the abundant flush away foreign protein of PBS.Press zymoplasm and shear capture agent box operation instructions, add gently mixing of endonuclease reaction liquid, make the two abundant mixing.In 20.5 ℃ of lower placements, constantly shake up gently reaction 12h.
It is as follows that enzyme is cut system:
The 10x zymoplasm is sheared capture buffer liquid 300 μ L
Zymoplasm (1: 100) the 1000 μ L of dilution
Target protein post adhesion protein amount (μ g)
1x PBS 1700μL
Vertical fixedly GST post was collected effluent liquid after reaction finished, and was the head product of purpose antigen.
The purpose antigen liquid of collecting is joined in the dialysis tubing of handling well, and dialyzate is 1 * PBS (pH7.4), and every 2h changes dialyzate once.Collect the good purpose antigen of purifying, the enzyme of SDS-PAGE electrophoresis detection fusion rotein is cut and purification effect.
The indirect ELISA detection method of embodiment 6 South Africa II type foot and mouth disease viruses
1, ELISA related solution prescription
Coated damping fluid (0.1mol/L carbonate buffer solution pH9.6): Na
2CO
33.18g, NaHCO
35.86g adding distil water is to 1000mL.
Lavation buffer solution (0.01mol/L PBST pH7.4 contains 0.05%Tween-20): Na
2HPO
412H
2O 2.91g, NaH
2PO
42H
2O 0.30g, NaCl 8.50g, Tween-200.5mL, adding distil water is to 1000mL.
Confining liquid: gelatin 1g is dissolved among the 100mL PBS.
Diluent: chicken ovalbumin (OVA) 0.1g adds lavation buffer solution to 100mL.
Substrate buffer solution (0.05mol/L phosphoric acid-citric acid pH5.0): 0.2mol/LNa
2HPO
4(28.4g/L) 25.7mL, 0.1mol/L citric acid (19.2g/L) 24.3mL, adding distil water is to 100mL.
TMB uses liquid: TMB (10mg/mL is dissolved in dimethyl formamide DMF) 150 μ L, substrate buffer solution 10mL, H
2O
26 μ L.
Stop buffer (1mol/L H
2SO
4): distilled water 578.3mL dropwise adds the vitriol oil (98%) 217.7mL.
2, step and the criterion of South Africa II type foot and mouth disease indirect ELISA detection method
(1) antigen coated concentration and serum optimum dilution degree determines
A, envelope antigen: with coated damping fluid (pH9.6Na
2CO
3-NaHCO
3) purpose antigen is diluted to respectively 0.1 μ g/ml, 0.2 μ g/ml, 0.4 μ g/ml, 0.8 μ g/m, 1.6 μ g/ml, 3.2 μ g/ml, 6.4 μ g/ml, 12.8 μ g/ml, every hole adds 100 μ L, 37 ℃ of 2h.
B, sealing: remove coating buffer, on paper, cramp gently to suck residual liquid, add lavation buffer solution (PBST) 300 μ L/ holes, wash altogether 3 times; Add 1% gelatin, 200 μ L/ holes, 37 ℃ of sealing 2h.
C, washing: discard liquid in the plate after sealing is finished, with PBST washing 5 times.
D, add standard positive serum: dilution is 1: 50,1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400 respectively, with foetal calf serum as negative control sera, 100 μ L/ holes, 37 ℃, 1h.
E, washing: discard liquid in the plate, with PBST washing 5 times.
F, adding ELIAS secondary antibody: add the anti-ox IgG of HRP-rabbit of dilution in 1: 10000,100 μ L/ holes, 37 ℃, 1h.
G, add substrate solution colour developing: after PBST washing 5 times, on paper, buckle gently residual liquid in the dry plate, add freshly prepared TMB and use liquid, 100 μ L/ holes, 37 ℃ of lucifuges reaction 15min.
H, termination reaction: every hole adds 50 μ L 1M H
2SO
4Termination reaction detects OD under microplate reader
450Value.
Measure OD
450Value, the highest according to positive value (P)/feminine gender value (N), the minimum principle of negative value, the chessboard distribution determines that the coated concentration of optimality criterion antigen is 6.4 μ g/ml, the weaker concn that best serum detects is 1: 200, as shown in Table 1 and Table 2.
Definite (OD of the best antigen coated concentration of table 1 and best serum dilution
450)
Definite (P/N) of the suitableeest coated concentration of table 2 antigen and serum optimal dilution
(2) the anti-ox extent of dilution of best HRP-rabbit is groped
Detect with the best coated concentration of antigen and best serum diluting multiple, the anti-ox of HRP-rabbit is done respectively dilution in 1: 1000,1: 2000,1: 4000,1: 8000,1: 16000,1: 32000, carry out indirect ELISA by above method and detect.According to OD
450Value, maximum according to positive value/feminine gender value, determine that the extent of dilution of the best anti-ox of HRP-rabbit is 1: 4000, as shown in table 3.
The best ELIAS secondary antibody Cigarette dilution detection of table 3 result
(3) negative threshold value determines
Choose at random 31 parts and detect through commercialization ELISA detection kit and to be defined as negative serum sample and to carry out ELISA and measure, measurement result is carried out variance analysis, and set threshold value (Q) according to literature method: the average OD of Q=negative sample
450Value+3SD (SD is standard variance).S (sample OD450)＞Q is judged to the positive; S＜Q is judged to feminine gender; In order to reduce false positive or false negative result, threshold value is added or deducts the scope of 1 standard deviation as a suspicious interval, namely during (Q-SD)＜S＜(Q+SD), be judged to suspicious.
The criterion definite according to aforesaid method: OD
450＞0.562 o'clock, positive; OD
450Be suspicious between 0.502～0.622, the result between suspicious interval needs duplicate detection, still is the suspicious positive that then is judged to such as the duplicate detection result; OD
450＜0.502 o'clock, negative, as shown in table 4.
Table 4 negative sample detected result (OD
450)
(4) specific detection
Indirect ELISA method by above foundation detects SAT I, SATIII, O, A, C and Asia I type FMDV standard positive serum, establishes SAT II type FMDV serum as positive control, and OD is read in the negative contrast of foetal calf serum
450, statistic data, through the SPSS11.5 software analysis, SAT II standard positive serum and other hypotype standard positive serum and negative serum difference is (P＜0.01) extremely significantly, and other hypotype serum OD
450All less than 0.502, show the FMDV antibody of SAT II serotype and the FMDV antibody no cross reaction of other serotype, such as table 5 not.
Table 5 indirect ELISA specific test result (OD
450)
Formation and the preparation method of embodiment 7 South Africa many epitopes of II type foot and mouth disease virus VP1～VP3 albumen envelope antigen test kits
1, many epitopes of South Africa II type foot and mouth disease virus VP1～VP3 albumen envelope antigen test kit comprises: 2 of antigen enzyme plates, 10 * lavation buffer solution 50mL, negative and positive serum each 500 μ L, substrate buffer solution 50mL, stop buffer 25mL, chicken ovalbumin 0.1g, anti-ox two anti-10 μ L, TMB (10mg/mL is dissolved in dimethyl formamide DMF) the 500 μ L of HRP-rabbit.
2, ELISA related solution prescription
Lavation buffer solution: get 10 * lavation buffer solution 10mL adding 90mL distilled water diluting and become 1 * lavation buffer solution.
Diluent: chicken ovalbumin (OVA) 0.01g adds lavation buffer solution to 10mL.
Two anti-working fluids: anti-ox two anti-the carrying out of HRP-rabbit are diluted at 1: 4000 with diluent.
TMB uses liquid: TMB (10mg/mL) 150 μ L, substrate buffer solution 10mL, H2O26 μ L.
3, the preparation of antigen enzyme plate
Many epitopes VP1 of above-described embodiment 5 preparation～VP3 albumen is diluted to 6.4 μ g/mL with coated damping fluid, join in the enzyme plate by 100 μ L/ holes, put 37 ℃ of 2h after, abandon coating buffer, add lavation buffer solution (PBST) 300 μ L/ holes, wash altogether 3 times; Add confining liquid (200 μ L/ hole) in 37 ℃ of sealing 2h; Abandon confining liquid, with PBST washing 5 times, air-dry; Enzyme plate is put into special-purpose Fresco Bag, add a pouch siccative, vacuumize hot-press sealed.
The Preliminary Applications of embodiment 8 indirect ELISA methods in clinical SAT II FMDV sample detection
Take out test kit from 4 ℃ of refrigerators, each component of balance is to room temperature.1 * the washings, diluent, two anti-working fluids, the TMB that press the step preparation appropriate amount among the embodiment 7 use liquid.39 parts of stochastic samplings are carried out ELISA and are detected from the many batches of domestic clinical samples that preserve in institute of China inspection section moving inspection laboratory, investigate the infection conditions of domestic South Africa type FMDV.Serum to be checked and positive serum are joined in the enzyme plate (100 μ L/ hole) after by dilution in 1: 200 with diluent put 37 ℃ of 1h.Discard liquid in the plate, with 1 * washings detersive enzyme target 5 times, each 300 μ L dry.Add two anti-working fluids, every hole 100 μ L put 37 ℃ of 1h.Discard liquid in the plate, with 1 * washings detersive enzyme target 5 times, each 300 μ L dry.Every hole adds TMB working fluid 100 μ L, 37 ℃ of lucifuge reaction 15min.Every hole adds 50 μ L stop buffers, and the light shaking termination reaction is put microplate reader, OD
450Survey light absorption value.Judging criterion: OD
450＞0.562 o'clock, positive; OD
450Be suspicious between 0.502～0.622, the result between suspicious interval needs duplicate detection, still is the suspicious positive that then is judged to such as the duplicate detection result; OD
450＜0.502 o'clock, negative.The detected result demonstration, in 39 duplicate samples, OD
450All less than 0.502, can be judged to feminine gender, as shown in table 6.
Detected result (the OD of the clinical serum sample of table 6
450)
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
＜110〉China Inst. of Quarantine Inspection Sciences
＜120〉the many epitope proteins of South Africa II type foot and mouth disease, its preparation method and application
<130>KHP10112127.5
<160>4
<170>PatentIn version 3.5
<210>1
<211>112
<212>PRT
＜213〉artificial sequence
<400>1
Arg Phe Phe Lys Glu Lys Leu Phe Asp Trp Thr Ser Asp Lys Pro Phe
1 5 10 15
Pro Ser Leu Gly Val Asn Arg His Asp Gln Gly Lys Arg His Gln Ala
20 25 30
Trp Ser Pro Ser Asp Gly Lys Pro Tyr Val Val Thr Lys Asn Asn Gly
35 40 45
Asp Lys Val Met Gly Gly Gly Gly Ser Tyr Asn Gly Glu Cys Lys Tyr
50 55 60
Thr Gln Thr Ser Thr Ala Ile Arg Gly Asp Arg Glu Val Leu Ala Gln
65 70 75 80
Lys Tyr Ser Ser Ala Lys His Ser Leu Pro Gly Gly Gly Gly Ser His
85 90 95
Gln Ser Arg Asp Arg Phe Asp Ala Pro Ile Gly Val Glu Lys Gln Leu
100 105 110
<210>2
<211>349
<212>DNA
＜213〉artificial sequence
<400>2
ggatcccgct tctttaagga aaagctgttt gattggacca gcgataaacc gtttccgagc 60
ctgggtgtga accgtcatga tcagggcaag cgtcatcagg cgtggagccc gagcgatggt 120
aaaccgtatg tggtgaccaa gaacaacggc gataaagtga tgggtggcgg tggcagctat 180
aacggtgaat gcaagtatac ccagaccagc accgcgattc gtggcgatcg tgaagtgctg 240
gcgcagaaat atagcagcgc gaagcatagc ctgccgggcg gtggcggtag ccatcagagc 300
cgtgatcgtt ttgatgcgcc gattggtgtg gaaaaacagc tgggtcgac 349
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<400>3
ggatcccgct tctttaagga 20
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<400>4
gtcgacccag ctgtttttcc a 21
Claims (8)
1. many epitopes of south Africa II type foot-and-mouth disease virus VP1～VP3 albumen is characterized in that, its aminoacid sequence is shown in SEQ ID No:1.
2. the gene of the viral many epitopes VP1 of the south Africa II type foot-and-mouth disease claimed in claim 1 of encoding～VP3 albumen, its nucleotide sequence is shown in SEQ ID NO:2.
3. the expression vector that contains the described nucleotide sequence of claim 2.
4. expression vector as claimed in claim 3, its carrier that sets out is pGEX-6P-1.
5. the recombinant expressed bacterium that contains the described nucleotide sequence of claim 2.
6. recombinant expressed bacterium as claimed in claim 5, its starting strain is BL21(DE3) plysS.
7. method for preparing the described many epitopes VP1 of claim 1～VP3 albumen, it comprises the steps:
1) dna fragmentation shown in the SEQ ID NO:2 is connected among the carrier pMD-19T, behind BamHI and SalI double digestion, is connected structure with the pGEX-6P-1 of the same double digestion of process and obtains expression vector pGEX-VP1～VP3;
2) the expression vector pGEX-VP1～VP3 with step 1) is converted into e. coli bl21 (DE3) plysS competent cell, screening positive clone and abduction delivering recombinant protein;
3) enzyme is cut and purification step 2) recombinant protein.
8. test kit that contains the described many epitopes VP1 of claim 1～VP3 albumen for detection of south Africa II type foot-and-mouth disease virus.
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| CN109799341A (en) * | 2018-12-07 | 2019-05-24 | 中国农业科学院兰州兽医研究所 | A kind of kit and preparation method detecting 2 type antibodies against foot-and-mouth disease virus of South Africa |
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| Title |
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| Ming Yang 等.Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus.《Veterinary Immunology and Immunopathology》.2007,第115卷第126－134页. * |
| 李雅静等.南非Ⅱ型***病毒抗原表位的筛选及抗原性检测.《中国畜牧兽医学会2008年学术年会暨第六届全国畜牧兽医青年科技工作者学术研讨会论文集》.2008, * |
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