Immune purification method for foot-and-mouth disease in large-scale pig farm

Abstract

The invention provides a method for purifying foot-and-mouth disease based on a method for detecting suspected positive foot-and-mouth disease virus nucleic acid, and aims to thoroughly remove foot-and-mouth disease pathogens in a group and in the environment. According to the provided method, an ELISA kit is used for detection, the kit comprises a monoclonal antibody, the amino acid sequence of a heavy chain of the monoclonal antibody is SEQ ID NO: 7, and the amino acid sequence of a light chain of the monoclonal antibody is SEQ ID NO: 9. The method comprises the following steps: enhancing immunization of pigs with unqualified antibodies, detecting foot-and-mouth disease neutralizing antibodies and VP1 antibodies after immunization, and eliminating immune antibody negative and VP1 antibody positive pigs. The ELISA kit provided by the invention is strong in stability and good in sensitivity, and when the kit is used for checking in a large-scale pig farm, the purification of the foot-and-mouth disease can be effectively realized. The purification method provided by the invention is high in accuracy and easy to popularize and apply.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of veterinary medicine, and particularly relates to a method for immunizing and purifying foot and mouth disease in a large-scale pig farm. BACKGROUND

[0002] Foot and Mouth Disease (FMD) is an acute, hot and highly contagious infectious disease of odd-toed animals caused by a small RNA virus of the Foot and Mouth Disease Virus. Clinically, blisters and ulcers occur in the oral mucosa, nose, hooves and breast skin of pigs. The incidence of swine foot and mouth disease is high, the transmission is fast, the epidemic area is large, and it can cause a large number of deaths in piglets. Different ages of pigs have different degrees of susceptibility, generally the younger the piglets, the higher the incidence, the more severe the disease, and the higher the mortality. Swine foot and mouth disease often occurs in late autumn, winter and early spring, especially in spring, but in large pig farms and pig storage warehouses, it can occur all year round; livestock products, people, animals, transportation tools, etc. are all transmission media of the disease. Antigenic difference of vaccine virus: FMDV can be divided into O, A, Asia I and other 7 serotypes, and there is no cross immunity between serotypes. Each serotype has many subtypes, and genetic variation between different strains within the type can cause antigenic difference. Swine foot and mouth disease is mainly O type and A type. Among them, O type is the most infected.

[0003] Foot and Mouth Disease is a reportable animal infectious disease stipulated by the World Organisation for Animal Health (WOAH), a class of epidemic diseases stipulated by the Animal Epidemic Prevention Law of the People's Republic of China, a class of infectious diseases that must be inspected for imported animals, and a class of animal diseases that must be prevented and treated in the National Medium and Long-Term Animal Disease Prevention and Control Plan (2012-2020). The country also lists the foot and mouth disease virus as a class of animal pathogenic microorganisms. The outbreak and prevalence of the disease often have a significant negative impact on the livestock production, animal-derived food supply, international trade and foreign political relations of a country.

[0004] The technical route for preventing and controlling foot-and-mouth disease in China is "compulsory immunization, pathogen elimination, herd purification, and basic eradication", and the comprehensive prevention and control measures of "prevention first, combination of immunization and culling" are adhered to. The method for preventing and controlling and eradicating foot-and-mouth disease outbreaks in the prior art mainly uses foot-and-mouth disease O type and A type bivalent 3B protein epitope deletion inactivated marker vaccine and foot-and-mouth disease VP1 blocking ELISA detection kit, adopts the method of immunization + detection + elimination, that is, after immunization with the deletion inactivated marker vaccine, the VP1 blocking ELISA detection kit is used for detection, the pig herd with positive detection is eliminated, the positive rate is gradually reduced, and finally the foot-and-mouth disease is purified. The WOAH manual (code) stipulates that the qualified foot-and-mouth disease inactivated vaccine has no VP1 antibody after 3 times of immunization, but experience analysis from a large number of detections shows that the VP1 antibody positive rate of part of the livestock immunized more than 3 times accounts for 11%-15%, and the VP1 antibody positive rate of the livestock immunized 3 times or less accounts for 3%-7%. In this way, it is impossible to distinguish between infected and immunized animals, eliminate positive animals, establish a healthy negative herd, and achieve effective prevention and control and purification of foot-and-mouth disease. Therefore, a method for purifying foot-and-mouth disease in a pig farm with positive foot-and-mouth disease virus infection is needed. SUMMARY

[0005] The purpose of the present application is to provide a method for purifying foot-and-mouth disease based on a foot-and-mouth disease virus nucleic acid suspected positive detection method, and the purpose is to completely eliminate foot-and-mouth disease virus.

[0006] The present application first provides a monoclonal antibody prepared using VP1 protein of foot-and-mouth disease virus as an antigen; the monoclonal antibody has the amino acid sequences of CDR of the heavy chain variable region as follows: GFTFSSYAMSIS (SEQ ID NO: 1), SAISGSGGSTYYADSRR (SEQ ID NO: 2), EGGYFDYWG (SEQ ID NO: 3), and the amino acid sequences of CDR of the light chain variable region as follows: QGDRVSSILAW (SEQ ID NO: 4), ASSLQWV (SEQ ID NO: 5), and QQYNSYKVE (SEQ ID NO: 6), Furthermore, the amino acid sequence of the heavy chain is as follows: GTLVVESGGGGRLLVQPAPGGSCAASGFTFSSYAMSISQKGLWVKGEWFTVSAISGSGGSTYYADSRRDNSKNTLYLQMNSLTAVYYCAREGGYFDYWGQVRAEDSSVRLSTEVQ (SEQ ID NO: 7); The sequence of the nucleotide fragment for encoding the heavy chain is as follows: GGTACTTTAGTTGTCGAATCTGGCGGAGGGGGTCGTTTGCTTGTACAACCTGCTCCCGGCGGATCCTGTGCCGCATCAGGGTTTACCTTCTCGAGTTATGCGATGAGCATTTCTCAGAAAGGTCTCTGGGTGAAGGGCGAGTGGTTTACAGTTTCCGCTATCTCAGGATCGGGGGGTAGTACGTACTATGCCGATAGCCGCCGAGACAATTCTAAAAACACTCTATACCTGCAAATGAATTCCTTAACCGCAGTCTATTACTGCGCGCGGGAAGGCGGATATTTCGATTACTGGGGGCAGGTAAGAGCTGAGGACTCATCGGTGAGGTTGAGTACAGAAGTTCAA (SEQ ID NO: 8); The amino acid sequence of a light chain is as follows: DQSPLQPEDIGKSVTSLSMTQASVTITCRGQGDRVSSILAWYSQKPAKAPKASSLQWVPSRFLIYSGSGSGSGTDFTLTISSTTYYAPYTFGQGCQQYNSYKVEIKSLGQAQLAA (SEQ ID NO: 9); The corresponding nucleotide sequence thereof is as follows: GACCAGTCTCCGCTGCAGCCCGAGGACATCGGCAAATCGGTGACCAGCCTGAGCATGACCCAGGCCAGCGTGACCATCACCTGCCGGGGCCAAGGCGACAGGGTGTCCAGCATCCTGGCTTGGTACAGCCAGAAGCCCGCCAAGGCCCCCAAGGCCAGCAGCCTGCAGTGGGTGCCCAGCCGGTTCCTGATCTACAGCGGCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCACCACCTACTACGCGCCCTACACCTTCGGCCAGGGCTGCCAGCAGTACAACAGCTACAAGGTGGAGATCAAGAGCCTGGGCCAGGCCCAGCTGGCCGCC (SEQ ID NO: 10).

[0007] The application further provides an application of the VP1 monoclonal antibody, which is to use the monoclonal antibody to prepare an ELISA kit for detecting foot-and-mouth disease antigens. In another aspect, the application provides a method for purifying foot-and-mouth disease in a large-scale pig farm, which comprises the following steps: detecting foot-and-mouth disease antigens by using the prepared ELISA kit, strengthening immunization for the pig population with unqualified antibodies, and detecting foot-and-mouth disease neutralizing antibodies and VP1 antibodies after immunization, and eliminating the pig population with negative immunization antibodies and positive VP1 antibodies.

[0008] The ELISA kit provided by the application has high stability and good sensitivity, and the use of the kit in the large-scale pig farm can effectively realize the purification of foot-and-mouth disease. The purification method provided by the application has high accuracy and is easy to popularize and apply. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 : SDS-PAGE method for detecting the purity of monoclonal antibody protein result graph; Figure 2 : Western blot method for detecting target protein electrophoresis graph; Figure 3 : Immunoblotting analysis of the reaction characteristics of monoclonal antibody; Figure 4 : IFA method for determining the reaction characteristics of monoclonal antibody 3A8 and FMDV, wherein A is FMDV and B is a control group; Figure 5 : Pig farm foot-and-mouth disease purification flow chart. DETAILED DESCRIPTION

[0010] The application will be described in detail below in combination with examples and drawings.

[0011] Example 1: Preparation of VP1 monoclonal antibody 1. Primer design and expression and purification of VP1 protein Based on the VP1 gene sequence of porcine foot-and-mouth disease virus (FMDV) (using the genome of O-type FMDV strain as a template), PCR amplification primers are designed as follows: Forward primer: 5'-CGCGGATCCATGGCGGTGGTGACCG-3', containing a BamH I restriction site; Reverse primer: 5'-CCGCTCGAGTTAGTGTTGCTGTGCTTG-3', containing a Xho I restriction site. After PCR amplification, the VP1 gene fragment is cloned into a prokaryotic expression vector pET-28a(+) and transformed into E. coli BL21(DE3) competent cells.

[0012] Positive recombinant strains were cultured in LB medium at 37℃ to OD600=0.6~0.8, and then 0.5mM IPTG was added to induce expression at 16℃ for 16 hours. After ultrasonic disruption, the supernatant was collected by centrifugation, and the His-tagged VP1 protein was purified by Ni-NTA affinity chromatography. SDS-PAGE analysis showed that the VP1 protein was successfully purified Figure 1 , and Western blot verified that it specifically bound to FMDV positive serum Figure 2 .

[0013] 2. Preparation and screening of monoclonal antibodies (1) Mouse immunization The purified VP1 protein (50μg) was emulsified with Freund's complete adjuvant and subcutaneously immunized 6~8 week-old BALB / c mice (n=4). Two weeks later, the mice were boosted with Freund's incomplete adjuvant + 30μg VP1, and then boosted twice every 14 days. One week after the last immunization, the serum titer was detected, and the mouse with the highest titer was selected for spleen cell fusion.

[0014] (2) Hybridoma cell screening The spleen cells of the immunized mouse were fused with SP2 / 0 myeloma cells (1:2 ratio) under the action of PEG-1500, and the hybridoma cells were screened by HAT medium.

[0015] The hybridoma supernatant was detected by coating the ELISA plate with VP1 protein, and the positive wells with OD450 / 630 value >2.0 were subcloned. After three subcloning, a hybridoma cell line stably secreting anti-VP1 monoclonal antibody was obtained (named FMDV-VP1-3A8).

[0016] 3. Identification of monoclonal antibodies (1) Antibody sequence analysis The amino acid sequence of the heavy chain of the prepared monoclonal antibody is as follows: GTLVVESGGGGRLLVQPAPGGSCAASGFTFSSYAMSISQKGLWVKGEWFTVSAISGSGGSTYYADSRRDNSKNTLYLQMNSLTAVYYCAREGGYFDYWGQVRAEDSSVRLSTEVQ; the amino acid sequences of the variable region CDRs are GFTFSSYAMSIS, SAISGSGGSTYYADSRR, EGGYFDYWG, respectively, and the corresponding nucleotide sequences are GGGTTTACCTTCTCGAGTTATGCGATGAGCATTTCT, TCCGCTATCTCAGGATCGGGGGGTAGTACGTACTATGCCGATAGCCGCCGA, GAAGGCGGATATTTCGATTACTGGGGG; The sequence of the nucleotide fragment for encoding the heavy chain is as follows: GGTACTTTAGTTGTCGAATCTGGCGGAGGGGGTCGTTTGCTTGTACAACCTGCTCCCGGCGGATCCTGTGCCGCATCAGGGTTTACCTTCTCGAGTTATGCGATGAGCATTTCTCAGAAAGGTCTCTGGGTGAAGGGCGAGTGGTTTACAGTTTCCGCTATCTCAGGATCGGGGGGTAGTACGTACTATGCCGATAGCCGCCGAGACAATTCTAAAAACACTCTATACCTGCAAATGAATTCCTTAACCGCAGTCTATTACTGCGCGCGGGAAGGCGGATATTTCGATTACTGGGGGCAGGTAAGAGCTGAGGACTCATCGGTGAGGTTGAGTACAGAAGTTCAA; The amino acid sequence of the light chain is as follows (115 aa): DQSPLQPEDIGKSVTSLSMTQASVTITCRGQGDRVSSILAWYSQKPAKAPKASSLQWVPSRFLIYSGSGSGSGTDFTLTISSTTYYAPYTFGQGCQQYNSYKVEIKSLGQAQLAA, the amino acid sequences of the variable region CDRs are QGDRVSSILAW, ASSLQWV, QQYNSYKVE, respectively, and the corresponding nucleotide sequences are CAAGGCGACAGGGTGTCCAGCATCCTGGCTTGG, GCCAGCAGCCTGCAG TGG, CAGCAGTACAACAGCTACAAGGTGGAG. The sequence of the nucleotide fragment encoding the light chain is as follows: GACCAGTCTCCGCTGCAGCCCGAGGACATCGGCAAATCGGTGACCAGCCTGAGCATGACCCAGGCCAGCGTGACCATCACCTGCCGGGGCCAAGGCGACAGGGTGTCCAGCATCCTGGCTTGGTACAGCCAGAAGCCCGCCAAGGCCCCCAAGGCCAGCAGCCTGCAGTGGGT GCCCAGCCGGTTCCTGATCTACAGCGGCTCCCGGCTCCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCACCACCTACTACGCGCCCTACACCTTCGGCCAGGGCTGCCAGCAGTACAACAGCTACAAGGTGGAGATCAAGAGCCTGGGCCAGGCCCAGCTGGCCGCC.

[0017] Reaction feature verification Western blot analysis showed that the purified FMDV VP1 protein presented a single clear band ( Figure 3 ), confirming that the monoclonal antibody (3A8) can specifically recognize VP1 protein and has no cross-reaction. Further verification by indirect immunofluorescence (IFA) showed that the monoclonal antibody 3A8 showed a significant green fluorescence signal when it was combined with FMDV-infected BHK-21 cells ( Figure 4 ), located in the cytoplasm, consistent with the viral replication site; while the uninfected control group had no fluorescence at all, indicating that 3A8 is highly specific to FMDV.

[0018] Combined with the WB and IFA results, this antibody can not only recognize the denatured VP1 protein (linear epitope), but also bind to the conformational epitope of natural virus particles, and is suitable for laboratory diagnosis and neutralization mechanism research of FMDV.

[0019] Example 2: Establishment of blocking ELISA detection method 1. ELISA detection method (1) Antigen coating: Dilute the purified pET-28a(+)-VP1 recombinant protein to 10 pg / mL with PBS, add 100 μL / well to the enzyme-linked plate, and incubate at 4°C overnight.

[0020] (2) Blocking: Wash the plate with PBST 3 times (3-5) min / time, add 200 μL 5% skim milk powder / well, and block at 37°C for 2 h.

[0021] (3) Primary antibody incubation: repeat washing, add 100 μL / well of 1:200 diluted positive / negative serum, incubate at 37°C for 1.5 h, 3 replicates per group.

[0022] (4) Secondary antibody incubation: wash the plate again, add 100 μL / well of 1:5000 diluted HRP-labeled secondary antibody, incubate at 37°C for 1.5 h.

[0023] (5) TMB color development: after washing the plate, add 100 μL / well of color developing solution A and B, react at 37°C for 15 min.

[0024] 2. Specificity test The prepared three batches of FMDV VP1 blocking ELISA antibody detection kit were used to detect swine vesicular disease (SVDV), swine vesicular stomatitis (VESV), swine vesicular exanthema (VESV) positive serum, and Escherichia coli (BL2) (DE3) positive pig serum, FMDV positive and negative serum, and the specificity of the detection method was evaluated. The results showed that the blocking rate of the kit for FMDV positive serum was higher than 80%, which was antibody positive, and there was no cross reaction with other control serum and negative serum, and the blocking rate was less than 50%, indicating that the method had good specificity.

[0025] 3. Sensitivity test The prepared blocking ELISA antibody detection kit was used to test different dilution concentrations (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560) of FMDV strongly positive serum, positive serum and weakly positive serum, each sample was tested in triplicate, and the sensitivity of the method established by the application was evaluated. The results showed that the lowest dilution of FMDV strongly positive serum was 1:320, the lowest dilution of positive serum was 1:40, and the lowest dilution of weakly positive serum was 1:10.

[0026] 4. Reproducibility test The test samples included five known FMDV positive sera and five negative sera, and each sample was tested five times in duplicate. Through statistical analysis of the OD 450 nm value, the stability of the detection method was verified. The detection results of 5 hidden sera were negative, and the detection results of 5 positive sera were all antibody positive. Statistical analysis was performed on the measured intra-batch and inter-batch OD 450 nm values, the intra-batch CV was 1.21%~3.25%, the inter-batch CV was 1.40%~4.86%, and the CV was less than 5%, which had good reproducibility.

[0027] 5. Coincidence rate test The blocking ELISA method established in the present application was applied to conduct comparative test with the commercially available foot-and-mouth disease VP1 blocking ELISA antibody detection kit. The 150 serum samples were detected clinically, and the coincidence rate between the two methods was calculated. The positive coincidence rate of the two methods was 99.33%, the negative coincidence rate was 98.67%, and the total coincidence rate was 99%.

[0028] The above results prove that the foot-and-mouth disease VP1 blocking ELISA antigen detection method established in the present application has strong specificity, high sensitivity and is suitable for rapid detection of a large number of samples.

[0029] Example 3: Foot-and-mouth disease immune purification of a certain pig farm The foot-and-mouth disease immune purification of a certain pig farm was carried out, and the purification process and key steps are shown in Figure 5 Before and after purification, the blood of boars, sows, commercial pigs and reserve pigs in the pig farm was collected at a rate of 10% per year, and the foot-and-mouth disease serum positive rate was detected. The purification method is as follows: 1. Epidemiological and immune investigation of the pig farm 1) The blood of boars, sows, commercial pigs and reserve pigs in the pig farm was collected at a rate of 10%, and the serum was separated. The foot-and-mouth disease liquid phase blocking ELISA (LPB-ELISA) and the prepared VP1 blocking ELISA detection kit were used for detection to understand the qualified rate of foot-and-mouth disease protective antibodies and the proportion of wild virus infection in the pig population.

[0030] 2) Investigation of foot-and-mouth disease immune scheme and vaccine used in the pig farm.

[0031] 3) When the foot-and-mouth disease VP1 antibody of the pig population is positive, there is a risk of wild virus infection.

[0032] 4) When the foot-and-mouth disease LPB-ELISA antibody of the pig is ≥1:64, it is determined that the pig is immune qualified. When the number of pigs with foot-and-mouth disease LPB-ELISA antibody ≥1:64 reaches more than 90%, the group is immune qualified.

[0033] 2. Formulate the immune scheme 1) Select the foot-and-mouth disease O type and A type bivalent 3B protein epitope deletion inactivated marker vaccine.

[0034] 2) The breeding pig population is immunized with foot-and-mouth disease vaccine 3 times per year, 2 mL per pig each time; the fattening pig population is immunized with foot-and-mouth disease vaccine at 56 days and 84 days, 2 mL per pig each time; the reserve pigs are immunized with foot-and-mouth disease vaccine at 140 days and 210 days, 2 mL per pig each time.

[0035] 3. Formulate the purification scheme If the vaccine used by the pig farm is the foot-and-mouth disease O type, A type bivalent 3B protein epitope deletion inactivated marker vaccine, 4 weeks after the third immunization of the pig group, the pig group is sampled at a proportion of 10%, and the foot-and-mouth disease LPB-ELISA and the VP1 blocking ELISA detection kit prepared above are used for detection, the qualified rate of foot-and-mouth disease neutralizing antibody of the pig group is more than 90%, and the ABC antibody is negative, then directly enter the immune purification state; at the same time, the pig group with unqualified antibody is strengthened, and 4 weeks after immunization, the foot-and-mouth disease neutralizing antibody and the VP1 antibody are detected again, and the immunized antibody negative and VP1 antibody positive pigs are eliminated. If the vaccine used by the pig farm is not the foot-and-mouth disease O type, A type bivalent 3B protein epitope deletion inactivated marker vaccine, then it is replaced with the foot-and-mouth disease O type, A type bivalent 3B protein epitope deletion inactivated marker vaccine, and the above immunization scheme and purification scheme are used for operation.

[0036] 4. Establishing a negative backup group The backup pigs are sampled and detected one by one 2 weeks before entering the group, if the non-structural protein antibody is negative and the neutralizing antibody titer is more than 1:64, the pigs can enter the breeding pig group; if the antibody is unqualified, the pigs are supplemented and immunized once, and then detected again after 28 days, if qualified, the pigs can enter the breeding pig group, if still unqualified, the pigs are not suitable for breeding.

[0037] 5. Breeding pig group monitoring The breeding pig group is sampled according to the plan and proportion, foot-and-mouth disease VP1 blocking ELISA detection kit is used for monitoring wild virus infection, and the positive breeding pigs are directly eliminated. The boars are detected once every 6 months, and the sows are detected at a proportion of 10% every 4 months.

[0038] 6. Biological safety prevention and control: adhere to the whole-in and whole-out system, strictly control the flow of vehicles, materials and personnel, strengthen isolation, disinfection, partition management, one-way flow, adhere to the measures of killing rats every quarter, and avoid the spread and diffusion of foot-and-mouth disease.

[0039] Table 1: foot-and-mouth disease nucleic acid detection result table from 2020 to 2024

[0040] As can be seen from the results in Table 1, positive samples were detected in the serum of the pig group of the pig farm from 2020 to 2022, indicating that there was a suspected positive phenomenon in the pig group, and the foot-and-mouth disease nucleic acid of various pigs in 2023 and 2024 was negative, indicating that the foot-and-mouth disease virus in the pig group was purified, confirming that the method provided by the application to purify the foot-and-mouth disease virus in the large-scale pig farm is effective.

Claims

1. A monoclonal antibody, characterized in that The monoclonal antibody is prepared by using the VP1 protein of foot-and-mouth disease virus as an antigen.

2. The monoclonal antibody according to claim 1, wherein The amino acid sequences of the heavy chain variable regions of the monoclonal antibodies are SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and the amino acid sequences of the light chain variable regions are SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

3. The monoclonal antibody according to claim 1, wherein The amino acid sequence of the heavy chain is SEQ ID NO:

7.

4. The monoclonal antibody according to claim 3, wherein The heavy chain, the sequence of its encoding nucleotide fragment is SEQ ID NO:

8.

5. The monoclonal antibody according to claim 1, wherein The amino acid sequence of the light chain is SEQ ID NO:

9.

6. The monoclonal antibody according to claim 5, wherein The light chain, the sequence of its encoding nucleotide fragment is SEQ ID NO:

10.

7. Use of the monoclonal antibody according to claim 1 in preparing an ELISA kit.

8. An ELISA kit for detecting foot-and-mouth disease antigens, characterized in that: The ELISA kit contains the monoclonal antibody according to claim 1.

9. A method for purifying foot-and-mouth disease in large-scale pig farms, characterized in that: The method is to use the ELISA detection kit described in claim 8 to detect pig herds, strengthen immunization of pig herds with unqualified antibodies, detect foot-and-mouth disease neutralizing antibodies and VP1 antibodies after immunization, and eliminate pigs with negative immune antibodies and positive VP1 antibodies.

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