CN101979406B - South African type Ⅱ foot-and-mouth disease multi-antigen epitope protein, its preparation method and application - Google Patents

Abstract

The present invention provides South African type II foot-and-mouth disease virus multi-antigen epitope VP1-VP3 protein, and the prokaryotic expression plasmid pGEX-VP1-VP3 constructed by encoding the multi-antigen epitope protein nucleotide sequence and prokaryotic expression vector pGEX-6P-1, Transfer pGEX-VP1～VP3 into Escherichia coli BL21(DE3)plysS to obtain a recombinant expression strain, digest and purify the protein expressed by the recombinant expression strain, and use the purified expression protein to establish a specific enzyme-linked immunosorbent assay for South African type II foot-and-mouth disease (ELISA) detection method, which has the advantages of good specificity, strong sensitivity, fast, simple, reliable, etc., and is suitable for rapid detection of a large number of samples.

Description

South African type Ⅱ foot-and-mouth disease multi-antigen epitope protein, its preparation method and application

technical field

The invention relates to the technical field of genetic engineering, in particular to a South African type II foot-and-mouth disease multi-antigen epitope VP1-VP3 protein, its preparation method and application.

Background technique

Foot-and-mouth disease (FMD) is an acute, highly contagious infectious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV), and was once listed as the first class A disease by the World Organization for Animal Health (OIE). Foot-and-mouth disease is a worldwide epidemic infectious disease, and its outbreak has caused great harm to global human health and animal husbandry production.

FMDV is an icosahedral single-stranded positive-strand RNA virus with a genome length of about 8.5kb, containing a large open reading frame, which is divided into four regions: L, P1, P2 and P3, of which the P1 region encodes the coat of the virus Capsid proteins VP4, VP2, VP3 and VP1, each with 60 molecules of VP4, VP2, VP3 and VP1 constitute the viral capsid protein, and most of VP1 is exposed on the surface of the virus particle, which is the main component that determines the antigenicity of the virus and can induce infection Animals produce neutralizing antibodies, VP4 is mostly buried inside the virus, VP2 and VP3 are between VP4 and VP1, and the four structural polypeptides are all involved in the composition of immunogenicity. VP1-VP3 constitute the capsid protein subunit, and VP4 is located inside the virus particle. The P2 gene encodes nonstructural proteins of three viruses, 2A, 2B, and 2C. The P3 gene encodes the nonstructural proteins 3A, Vpg, 3Cpro and 3Dpol.

There are seven serotypes of FMD virus, namely O, A, C, SAT I, SAT II, SAT III and Asia I. These seven serotypes can be divided into two groups according to their homology, O, A, C and Asia I are the first group, SAT I, SAT II, and SAT III are the second group, and the homology of each type in the group reaches 60. % to 70%, but the homology between the two groups is only 25% to 40%, and there is no cross-reaction between the serotypes of FMD virus. Even within the same serotype, the antigenicity of different viruses is different, which gives the The quarantine and prevention and control have increased the difficulty.

Traditional FMD diagnosis and quarantine methods mainly include serology such as complement fixation test (CFT), indirect hemagglutination test (PHA), agar diffusion test (AGP), virus neutralization test (VNT), enzyme-linked immunosorbent assay (ELISA), etc. Methods: With the development of molecular biology and the application of RT-PCR technology and gene chip technology, the detection of foot-and-mouth disease virus is developing towards a more sensitive, specific, convenient and rapid direction. In 1952, Brooksby established CFT. The method combining virus isolation and proliferation with complement fixation test has been used in the diagnosis of foot-and-mouth disease for nearly 30 years. Although this technique is accurate and reliable, it is not sensitive enough, and some samples have anti-complement activity, which affects Detection effect. The indirect hemagglutination test uses red blood cells as the carrier. According to the characteristics of the antigen and antibody of the sensitized red blood cells, known antigens or antibodies can be used to detect unknown antibodies or antigens. At present, there are commercial FMD indirect hemagglutination kits, which are widely used in the detection of FMD antibodies. The agar diffusion test detects both antigens and antibodies. According to the principle that the antigen and antibody diffuse each other in the gel and produce a precipitation line at the optimum ratio, the corresponding antibody or antigen can be detected with a known antigen or antibody. Virus neutralization test (VNT) is also called serum neutralization test. VNT can not only identify antigens, but also quantify antibodies. It has type specificity. It is the most classic detection method of FMD and the gold standard for evaluating other methods. RT-PCR technology is used for the detection of foot-and-mouth disease virus, which has the advantages of high sensitivity, good specificity, rapidity, and accuracy. PCR products can also be used for sequencing to obtain more detailed epidemiological information. Evolution provides information.

ELISA detection of foot-and-mouth disease has the advantages of good specificity, strong sensitivity, rapidity, simplicity, reliability, etc., and it can be operated automatically, which is suitable for rapid detection of a large number of samples. One of the routine methods for foot and mouth disease. The traditional method for detecting serum antibodies to FMD is mainly the indirect ELISA method using FMDV cultured in BHK cells as the antigen. Although this method has high specificity and sensitivity, it is easy to cause the spread of the virus during the production process of the virus. Many safety hazards require a BSL-3 laboratory environment. For this reason, many researchers expressed the structural and non-structural proteins of FMDV in Escherichia coli, using them as detection antigens, and established a variety of indirect ELISA detection methods for FMDV.

Africa is a natural source of FMD. In history, South African Type II (SAT II) FMD has never spread across the equator. This has led some scholars to believe that South African FMD requires a special living environment in Africa. However, in 2000, the South African Type II FMD outbreak in Kuwait and Saudi Arabia made people discover that SAT II Type FMD can not only cross the equator, but also cross the Red Sea and reach the Asian continent. Especially with the development of the African economy and the increasingly frequent contact with the world market, the gradual opening up of the African continent will also spread the disease that originally settled in Africa to the rest of the world.

With the establishment of diplomatic relations and frequent trade exchanges between my country and African countries, my country is increasingly threatened by the South African type of foot-and-mouth disease, and the SAT II type is the most threatening to our country. The current research on foot-and-mouth disease in my country is limited to O, A, C, and Asia I types of foot-and-mouth disease virus, while the research on SAT type II, including detection technology and vaccines, is completely blank.

In the present invention, on the basis of combining SAT II type FMDV structural protein VP1, VP2 and VP3 epitope genes in series with flexible amino acids and performing fusion expression in Escherichia coli, the SAT II type foot-and-mouth disease virus specific Serum type detection method to meet the specific detection and monitoring requirements of related animals and products.

Contents of the invention

The purpose of the present invention is to provide a South African type II foot-and-mouth disease multi-antigen epitope VP1-VP3 protein.

Another object of the present invention is to provide a method for preparing the multi-antigen epitope protein.

Another object of the present invention is to provide the application of the multi-antigen epitope protein.

In order to realize the object of the present invention, the present invention provides a kind of South African type II foot-and-mouth disease virus multi-antigen epitope VP1～VP3 protein, it has the aminoacid sequence as shown in SEQ ID NO: 1 or this sequence is replaced, deleted or added one or several An amino acid sequence with equivalent functions formed by two amino acids.

The present invention also provides a gene encoding the above-mentioned South African type II foot-and-mouth disease virus multi-antigen epitope VP1-VP3 protein, the nucleotide sequence of which is shown in SEQ ID NO:2.

The present invention also provides an expression vector containing the above nucleotide sequence.

The starting vector of the aforementioned expression vector is pGEX-6P-1.

The present invention also provides recombinant expression bacteria containing the above nucleotide sequence.

The starting strain of the aforementioned recombinant expression bacteria is BL21(DE3)plysS.

The present invention also provides a preparation method of South African type II foot-and-mouth disease virus multi-antigen epitope VP1～VP3 protein, which comprises the following steps: 1) connecting the DNA fragment shown in SEQ ID NO: 2 into the carrier pMD-19T, passing through BamHI and After SalI double digestion, it was connected with pGEX-6P-1 that had undergone the same double digestion to construct the expression vector pGEX-VP1～VP3; 2) Transform the expression vector pGEX-VP1～VP3 in step 1) into Escherichia coli BL21 (DE3 ) plysS competent cells, screening positive clones and inducing the expression of recombinant protein; 3) digestion and purification of the recombinant protein in step 2).

The invention also provides the application of multiple antigen epitope VP1-VP3 proteins of South African type II foot-and-mouth disease virus in detecting South African type II foot-and-mouth disease virus.

The invention further provides a kit for detecting the South African type II foot-and-mouth disease virus, which contains the multi-antigen epitope VP1-VP3 protein coating antigen.

The advantages of the present invention are that, the multi-antigen epitope VP1-VP3 protein of South African type II foot-and-mouth disease virus is prepared by the method of the present invention, which has a high safety factor, is friendly to the environment, and the preparation method is simple and easy; and the kit of the present invention is used to detect South African type II foot-and-mouth disease Viruses have the advantages of good specificity, strong sensitivity, rapidity, simplicity, and reliability, and are suitable for rapid detection of a large number of samples.

Description of drawings

Fig. 1 is the construction flowchart of recombinant expression plasmid pGEX-VP1～VP2 of the present invention;

Figure 2 is a schematic diagram of the SDS-PAGE results of the expression product of the recombinant bacteria of the present invention, wherein, M is a protein marker, 1 is the induced expression of the empty vector bacteria, 2 is the uninduced control of the recombinant expression bacteria, 3 and 4 are the recombinant expression bacteria 3h after induction expressed fusion protein;

Fig. 3 is a schematic diagram of the detection results of Western Blot using anti-GST monoclonal antibody for the fusion protein of the present invention, wherein, M is a protein marker, 1 is a GST-VP1-VP3 fusion protein, and 2 is a GST carrier protein;

Fig. 4 is the detection result schematic diagram that fusion protein of the present invention carries out WesternBlot with SAT II type FMDV positive serum, wherein, M is protein label, and 1 is GST-VP1～VP3 fusion protein, and 2 is GST carrier protein;

Figure 5 is a schematic diagram of the enzymatic digestion and purification results of the multi-antigen epitope VP1-VP3 protein of South African type II foot-and-mouth disease virus of the present invention, wherein, M is a protein marker, 1 and 2 are blank controls, 3 is a liquid phase direct enzyme digestion product, 4 and 5 are pGEX-VP1-VP3 fusion protein, 6 is GST protein, and 7 is the target antigen after purification.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1 Construction of South African Type II Foot-and-Mouth Disease Virus Multi-antigen Epitope Prokaryotic Expression Vector pGEX-VP1～VP3

Use a flexible amino acid linker to connect six peptides with good antigenicity VP2(60-73), VP2(163-176), VP3(58-71), VP3(127-140), VP1(132-146), VP1(199 -211), chemically synthesized corresponding DNA fragment, its nucleotide sequence is as shown in SEQ ID NO: 2. Then, the above DNA fragments were connected into the cloning vector pMD-19T, and the pMD-19T-VP1～VP3 plasmids were double-digested with BamH I and Sal I, and the pGEX-6P-1 plasmid was subjected to the same double enzyme digestion, and glued The target fragments of the two were recovered and constructed into prokaryotic expression vectors pGEX-VP1-VP3 by ligation, as shown in FIG. 1 .

The cloning of embodiment 2 South African type II foot-and-mouth disease virus multi-antigen epitope gene VP1～VP3 DNA sequence

Extract the pGEX-VP1～VP3 plasmid, use the plasmid as a template, amplify the DNA sequence of the multiple antigenic epitopes VP1～VP3 of South African type II foot-and-mouth disease virus by PCR, and design primers P1, P2, P1 and P2 primers contain BamH I and Sal I restriction sites respectively, and the primer sequences are as follows:

CGCTTCTTTAAGGA-3’P1: 5'-

CGCTTCTTTAAGGA-3'

CCAGCTGTTTTTCCA-3’P2: 5'-

CCAGCTGTTTTTCCA-3'

Among them, the restriction sites of BamH I and Sal I are respectively indicated in italics.

The PCR reaction system was: 1 μL of recombinant plasmid, 2.5 μL of 10×PCR Buffer (Mg ²⁺ ), 2 μL of dNTP, 1 μL of P1 and P2 primers, 0.5 μL of rTaq DNA polymerase, and supplemented with sterilized ddH ₂ O to 25 μL. Reaction program: 94°C for 5min; 94°C for 1min, 50°C for 50s, 72°C for 1min, a total of 30 cycles; 72°C for 10min.

Enzyme digestion identification: Recombinant expression plasmids pGEX-VP1~VP3 were extracted, and restriction endonucleases BamH I and Sal I were used for enzyme digestion identification. The enzyme digestion reaction system was: plasmid DNA 2 μg, 10×T buffer 7.5 μL, BamH I 2 μL, 2 μL of Sal I, 50 μL of the system was supplemented with sterilized ddH ₂ O, mixed evenly and digested in a water bath at 37°C for 3 hours.

The recombinant expression strains that were positive in both PCR identification and enzyme digestion identification were sent to Beijing Nuosai Genome Research Center for sequencing.

Example 3 Construction of Recombinant Escherichia coli BL21(DE3)plysS/pGEX-VP1～VP3

Transform the identified recombinant expression vector pGEX-VP1～VP3 into Escherichia coli BL21(DE3)plysS competent cells, smear LB/ampicillin (Amp) plates, pick multiple colonies and put them in LB liquid medium for culture at 37°C After 12 hours, the expression was induced by isopropylthio-β-D-galactoside (IPTG), and then detected by SDS-PAGE and Western-blot. The results are shown in Figure 2 to Figure 5 . The recombinant Escherichia coli BL21 (DE3) plysS/pGEX-VP1 - VP3 capable of inducing and expressing South African type II foot-and-mouth disease virus multi-antigen epitope genes VP1 - VP3 proteins in Escherichia coli BL21 (DE3) was screened out.

The determination of embodiment 4 optimal inducer concentration and optimal induction time

Pick a single colony of recombinant Escherichia coli BL21(DE3)plysS/pGEX-VP1～VP3 into 3 mL of LB medium, add ampicillin (Amp) to a final concentration of 50 μg·mL ^-1 , and culture it on a shaker at 37°C for 12 hours. The bacterial solution was transferred to 3 mL of fresh 2×YT (containing 50 μg·mL ^-1 Amp) medium at a 1% inoculum amount, cultivated at 37°C for 200 r·min ^-1 until the OD ₆₀₀ was about 0.6-0.8, and the final IPTG at a concentration of 1 mmol·L ^-1 was induced to express at 37°C, and 1 mL of bacterial liquid was collected at 2h, 4h, 6h, and 8h after induction, and the bacterial cells were collected by centrifugation at 12000r·min ^-1 . Suspend in 50 μL pH7.4 PBS, add an equal amount of 2× loading buffer, treat in a boiling water bath for 10 min, and centrifuge the treated bacterial sample at room temperature for 3 min at 10,000 r·min ^-1 . Take 10 μL of the supernatant for SDS-PAGE, and set the uninduced bacterial liquid as a control. After electrophoresis, stain with Coomassie brilliant blue and decolorize with acetic acid to observe the results. According to the expression of the multi-antigen epitope protein, the optimal induction time was determined to be 6 hours.

After the induction time was determined, they were induced for 6 hours under the conditions of IPTG concentrations of 0.1, 0.5, 1.0 and 1.5 mmol·L ^-1 respectively, and 1 mL of bacterial liquid was collected, and the bacterial cells were collected by centrifugation. Bacterial samples were treated according to the above method and then subjected to SDS-PAGE to determine the optimal concentration for inducing protein expression. The optimum induction concentration of IPTG was determined to be 1.0mmol·L ^-1 according to the expression of multiple epitope proteins.

Example 5 Large-scale induced expression and purification of South African type II foot-and-mouth disease virus multi-antigen epitope VP1-VP3 protein

Pick a single colony of recombinant Escherichia coli BL21(DE3)plysS/pGEX-VP1～VP3 into 3 mL of LB medium, add ampicillin to a final concentration of 50 μg/mL, culture overnight on a shaker at 37°C, take 10 μL of the overnight bacterial solution the next day and add Into 30 mL of LB medium containing 50 μg/mL ampicillin, cultured on a shaker at 37°C until the OD ₆₀₀ was about 0.6-0.8, added the inducer IPTG to a final concentration of 1 mmol/L, and continued to induce culture for 6 hours.

Centrifuge 30mL of the induced expression bacterial solution (pGEX-VP1～VP3) at 12000r/min for 2min to collect the bacterial cells, add 3mL MagneGST bacteriolysis reagent, and resuspend the bacterial pellet. Add 300 μL of RNase-Free DNase I, and incubate the bacterial suspension on a shaker for 30 min at room temperature to fully lyse it. The lysate was mixed with glutathione Sepharose ^™ -4B (2ml homogenate) filler, and adsorbed on a shaking table at room temperature for 30min. Wash thoroughly with PBS to remove impurities. According to the operating instructions of the thrombin shear capture kit, add the enzyme digestion reaction solution and mix gently to make the two fully mixed. Place it at 20.5°C, shake gently constantly, and react for 12 hours.

The enzyme digestion system is as follows:

10x thrombin shear capture buffer 300μL

Diluted thrombin (1:100) 1000μL

Target protein Amount of protein adsorbed on column (μg)

1x PBS 1700μL

After the reaction, fix the GST column vertically and collect the effluent, which is the primary product of the target antigen.

The collected target antigen solution was added to the processed dialysis bag, the dialysis solution was 1×PBS (pH7.4), and the dialysis solution was changed every 2 hours. The purified target antigen was collected, and SDS-PAGE electrophoresis was used to detect the digestion and purification effects of the fusion protein.

The indirect ELISA detection method of embodiment 6 South African type II foot-and-mouth disease virus

1. ELISA related solution formula

Coating buffer (0.1mol/L carbonate buffer pH9.6): Na ₂ CO ₃ 3.18g, NaHCO ₃ 5.86g, add distilled water to 1000mL.

Wash buffer (0.01mol/L PBST pH7.4, containing 0.05% Tween-20): Na ₂ HPO ₄ ·12H ₂ O 2.91g, NaH ₂ PO ₄ ·2H ₂ O 0.30g, NaCl 8.50g, Tween-200.5 mL, add distilled water to 1000mL.

Blocking solution: Dissolve 1g gelatin in 100mL PBS.

Diluent: chicken ovalbumin (OVA) 0.1g, add washing buffer to 100mL.

Substrate buffer (0.05mol/L phosphoric acid-citric acid pH5.0): 0.2mol/L Na ₂ HPO ₄ (28.4g/L) 25.7mL, 0.1mol/L citric acid (19.2g/L) 24.3mL, add Distilled water to 100mL.

TMB solution: TMB (10 mg/mL, dissolved in dimethylformamide DMF) 150 μL, substrate buffer 10 mL, H ₂ O ₂ 6 μL.

Stop solution (1mol/L H ₂ SO ₄ ): 578.3 mL of distilled water, 217.7 mL of concentrated sulfuric acid (98%) was added dropwise.

2. Steps and criteria for the indirect ELISA detection method for type II foot-and-mouth disease in South Africa

(1) Determination of antigen coating concentration and optimal dilution of serum

A. Coating antigen: Dilute target antigen to 0.1μg/ml, 0.2μg/ml, 0.4μg/ml, 0.8μg/m, 1.6μg with coating buffer (pH9.6Na ₂ CO ₃ -NaHCO ₃ ) /ml, 3.2μg/ml, 6.4μg/ml, 12.8μg/ml, add 100μL to each well, 37℃ for 2h.

B. Sealing: Remove the coating solution, gently tap on the paper to absorb the residual liquid, add washing buffer (PBST) 300 μL/well, wash 3 times in total; add 1% gelatin 200 μL/well, 37 ° C for 2 hours .

C. Washing: Discard the liquid in the plate after sealing, and wash 5 times with PBST.

D. Add standard positive serum: dilute to 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 respectively, and use fetal bovine serum as negative control serum , 100 μL/well, 37°C, 1h.

E. Washing: discard the liquid in the plate, and wash 5 times with PBST.

F. Add enzyme-labeled secondary antibody: add 1:10000 diluted HRP-rabbit anti-bovine IgG, 100 μL/well, 37°C, 1h.

G. Add substrate solution for color development: After washing with PBST for 5 times, lightly pinch the remaining liquid in the dry plate on the paper, add freshly prepared TMB use solution, 100 μL/well, and react in the dark at 37°C for 15 minutes.

H. Stopping the reaction: Add 50 μL of 1M H ₂ SO ₄ to each well to stop the reaction, and detect the value of OD ₄₅₀ with a microplate reader.

The OD ₄₅₀ value was measured. According to the principle of the highest positive value (P)/negative value (N) and the smallest negative value, the checkerboard distribution method determined that the coating concentration of the best standard antigen was 6.4 μg/ml, and the dilution of the best serum test was The concentration is 1:200, as shown in Table 1 and Table 2.

Table 1 Determination of optimal antigen coating concentration and optimal serum dilution (OD ₄₅₀ )

Table 2 Determination of optimal coating concentration of antigen and optimal dilution of serum (P/N)

(2) Search for the best HRP-rabbit anti-bovine dilution

The detection was carried out with the optimal coating concentration of the antigen and the optimal serum dilution multiple, and the HRP-rabbit anti-bovine were diluted 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 respectively, according to The above method was used for indirect ELISA detection. According to the OD ₄₅₀ value, according to the maximum positive value/negative value, the optimal dilution ratio of HRP-rabbit anti-bovine was determined to be 1:4000, as shown in Table 3.

Table 3 The best enzyme-labeled secondary antibody dilution detection results

(3) Determination of negative cut-off value

Randomly select 31 serum samples determined to be negative by commercialized ELISA detection kits for ELISA determination, perform analysis of variance on the measurement results, and set the critical value (Q) according to the method in the literature: Q = average OD ₄₅₀ value of negative samples + 3SD (SD is standard deviation). S(sample OD450)>Q is judged as positive; S<Q is judged as negative; in order to reduce false positive or false negative results, the range of the critical value plus or minus 1 standard deviation is taken as a suspicious interval, that is (Q- SD)<S<(Q+SD), it is judged suspicious.

According to the judgment standard determined by the above method: when OD ₄₅₀ > 0.562, it is positive; when OD ₄₅₀ is between 0.502 and 0.622, it is suspicious, and the results in the suspicious range need to be tested repeatedly. If the repeated test results are still suspicious, it is judged positive ; When OD ₄₅₀ <0.502, it is negative, as shown in Table 4.

Table 4 Test results of negative samples (OD ₄₅₀ )

(4) Specific detection

Detect SAT I, SATIII, O, A, C and Asia I type FMDV standard positive serum according to the indirect ELISA method established above, set SAT II type FMDV serum as positive control, fetal bovine serum as negative control, read OD ₄₅₀ , and count Data, analyzed by SPSS11.5 software, the difference between SAT II standard positive serum and other subtype standard positive serum and negative serum is extremely significant (P<0.01), and other subtype serum OD ₄₅₀ is less than 0.502, indicating that the SAT II serotype The FMDV antibody has no cross-reaction with the FMDV antibody of other serotypes, as shown in Table 5.

Table 5 Indirect ELISA specific test results (OD ₄₅₀ )

Example 7 The composition and preparation method of the South African type II foot-and-mouth disease virus multi-antigen epitope VP1～VP3 protein-coated antigen kit

1. South African type II foot-and-mouth disease virus multi-antigen epitope VP1-VP3 protein-coated antigen kit includes: 2 antigen microtiter plates, 50 mL of 10× washing buffer, 500 μL of positive and negative serum, 50 mL of substrate buffer, and 25 mL of stop solution , chicken ovalbumin 0.1 g, HRP-rabbit anti-bovine secondary antibody 10 μL, TMB (10 mg/mL, dissolved in dimethylformamide DMF) 500 μL.

2. ELISA related solution formula

Washing buffer: Take 10 mL of 10× washing buffer and add 90 mL of distilled water to dilute to 1× washing buffer.

Diluent: chicken ovalbumin (OVA) 0.01g, add washing buffer to 10mL.

Secondary antibody working solution: Dilute the HRP-rabbit anti-bovine secondary antibody 1:4000 with diluent.

TMB use solution: TMB (10mg/mL) 150μL, substrate buffer 10mL, H2O26μL.

3. Preparation of antigen microtiter plate

Dilute the multi-antigen epitope VP1-VP3 proteins prepared in the above-mentioned Example 5 to 6.4 μg/mL with coating buffer, add 100 μL/well to the microtiter plate, place at 37°C for 2 hours, discard the coating solution, and add Washing buffer (PBST) 300 μL/well, wash 3 times in total; add blocking solution (200 μL/well) and block at 37°C for 2 hours; discard blocking solution, wash 5 times with PBST, and air dry; put the microplate in a special tin foil bag , add a small bag of desiccant, vacuumize, heat seal.

Example 8 Preliminary application of indirect ELISA method in clinical SAT II FMDV sample detection

Remove the kit from the 4°C refrigerator and equilibrate the components to room temperature. According to the steps in Example 7, an appropriate amount of 1× washing solution, dilution solution, secondary antibody working solution, and TMB use solution were prepared. A random sampling of 39 domestic clinical samples from multiple batches of domestic clinical samples kept in the animal testing laboratory of the Chinese Academy of Inspection and Quarantine was carried out for ELISA testing to investigate the infection of South African FMDV in China. The serum to be tested and the positive serum were diluted 1:200 with diluent and added to the microtiter plate (100 μL/well) and placed at 37°C for 1h. Discard the liquid in the plate, wash the microtiter plate 5 times with 1× washing solution, 300 μL each time, and shake dry. Add secondary antibody working solution, 100 μL per well, and place at 37°C for 1h. Discard the liquid in the plate, wash the microtiter plate 5 times with 1× washing solution, 300 μL each time, and shake dry. Add 100 μL of TMB working solution to each well, and react at 37°C for 15 minutes in the dark. Add 50 μL of stop solution to each well, shake gently to stop the reaction, set up a microplate reader, and measure the absorbance at OD ₄₅₀ . Judgment criteria: when OD ₄₅₀ > 0.562, it is positive; when OD ₄₅₀ is between 0.502 and 0.622, it is suspicious, and the results in the suspicious range need to be tested repeatedly. If the repeated test results are still suspicious, it is judged positive; OD ₄₅₀ < 0.502 , it is negative. The test results showed that among the 39 samples, the OD ₄₅₀ was less than 0.502, which could be judged as negative, as shown in Table 6.

Table 6 Detection results of clinical serum samples (OD ₄₅₀ )

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

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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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