Foot-and-mouth disease virus type O specific neutralizing swine monoclonal antibody and application thereof

Abstract

The invention discloses a neutralizing swine monoclonal antibody pO18-40 and a neutralizing swine monoclonal antibody pO18-43 for foot and mouth disease virus type O. The amino acid sequences of a heavy chain variable region (VH) and a light chain variable region (VL) of the antibody pO18-40 are respectively as shown in SEQ ID No. 1 and SEQ ID No. 2; the amino acid sequences of VH and VL of the antibody pO18-43 are respectively as shown in SEQ ID No. 3 and SEQ ID No. 4. The antibody obtained by the invention is a full-swine-source antibody, can specifically neutralize the classical strain of the O-type foot-and-mouth disease virus, and can clearly distinguish the classical strain of the O / Cathay topological type from the variant strain of the O / Cathay topological type. A key antigen epitope recognized by the antibody is located at the 149th amino acid of a VP1 protein G-H ring, and the site is a key site of O / Cathay strain antigen variation and vaccine immune protection. The antibody provided by the invention provides an important tool and theoretical basis for serological detection of O-type FMDV, vaccine immune effect evaluation and optimal design of broad-spectrum vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to porcine monoclonal antibodies against type O foot-and-mouth disease virus and their applications. Background Technology

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease caused by the foot-and-mouth disease virus (FMDV) in cloven-hoofed animals such as pigs, cattle, and sheep. The World Organisation for Animal Health (WOAH) lists it as a notifiable animal disease, and my country has designated it as a priority major animal disease for prevention and control.

[0003] FMDV comprises seven serotypes, with type O being the most widespread, exhibiting 11 serotypes globally. The virus is prone to mutation, making effective control difficult. Currently, type O FMDV is the most serious serotype threatening my country's livestock industry. In recent years, three serotypes and four lineages (Cathay, ME-SA / PanAsia, ME-SA / Ind-2001, and SEA / Mya98) have been circulating concurrently. Recent studies indicate a declining trend in genetic diversity for SEA and ME-SA serotypes, while the evolutionary rate of the Cathay serotype has accelerated, with a significant increase in genetic diversity over the past decade, suggesting an expanding and spreading trend of the Cathay serotype in Asia.

[0004] Crucially, the O / Cathay variant shows a significantly reduced match with existing O-type vaccine strains, suggesting a structural mutation at key antigenic sites in this topological variant. The molecular basis of this antigenic structural mutation remains unclear, resulting in a lack of theoretical support for the development and optimization of broad-spectrum O-type vaccines and hindering precise control of O-type foot-and-mouth disease. Furthermore, existing foot-and-mouth disease virus detection reagents are mostly prepared from antibodies not derived from natural hosts, exhibiting low matching with the immune response characteristics of natural hosts. This makes it difficult to accurately distinguish between classic and variant strains, and the specificity and sensitivity of the detection need improvement. Summary of the Invention

[0005] The purpose of this invention is to provide porcine monoclonal antibodies pO18-40 and pO18-43, which are neutralizing antibodies against type O foot-and-mouth disease virus. These antibodies are derived from the natural host, pigs, and can specifically recognize key antigenic sites of type O FMDV. They exhibit good neutralizing activity against classic strains and can clearly distinguish between classic strains and variant strains.

[0006] Another objective of this invention is to provide applications for the aforementioned porcine monoclonal antibodies, offering new tools and theoretical basis for the detection of type O foot-and-mouth disease virus, the screening and optimization design of vaccine strains, and achieving precise prevention and control of foot-and-mouth disease.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a porcine monoclonal antibody pO18-40 that neutralizes type O foot-and-mouth disease virus, the amino acid sequence of its heavy chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of its light chain variable region is shown in SEQ ID No. 2.

[0008] pO18-40 heavy chain variable region (SEQ ID No. 1): EEKVVESGGGQVRPGESLRLSCLAEGVTFSRHSVRWVRQSPGKGLEFVIYMFNAVDSVDIADYVKGRFTVSRDNAQNTAYLQMTKLRVEDTGRYFCLIGNKWFGPGVEVVVSS; pO18-40 light chain variable region (SEQ ID No. 2): AIVLTQSPLYLSVTPGEPASISCKSTKSVLDTDGTTVLNWYLQKPGQAPQRVIWSAIYRETGVPDRFTGSGSGTDFTLYISRVEAEDVGVYYCQQSRESTSFGAGTKLDLK.

[0009] The present invention also provides a porcine monoclonal antibody pO18-43 that neutralizes type O foot-and-mouth disease virus, the amino acid sequence of its heavy chain variable region is shown in SEQ ID No. 3, and the amino acid sequence of its light chain variable region is shown in SEQ ID No. 4.

[0010] pO18-43 heavy chain variable region (SEQ ID No. 3): EEKVVESGGGLVQPGGSLRLSCVGSGYMFSKYGMTWVRHSQRKGLEWLAGIDNFESTDYAASVKGRFTLWRDNSQNTAYLEMSFLGVEDTARYYCGRGFGVPYIWGPGVEVVVSS; pO18-43 light chain variable region (SEQ ID No. 4): AVELTQTPVSLSVSPGEPASISCRSSQSLLHSDGRNYLTWYRIKPGQSPQRLVYYGTYRDPEIPDRFTGSESGTDFTLKISRVEAEDAGVYYCLQSKEPPYEFGAGNTLDLK.

[0011] The monoclonal antibodies pO18-40 and pO18-43 are both type O FMDV-specific neutralizing antibodies, exhibiting good neutralizing activity (IC50) against the classical O / Cathay strain O / HN / CHA / 93, the type O PanAsia strain O / Xizang / 99, and the Mya98 strain O / GSLX / 2010. 50 ≤50 μg·mL⁻¹) and binding capacity; however, it has no neutralizing activity (IC50) against O / Cathay topological variants (including O / SCGH / CHA / 2016, O / 18074, O / GD / 2021, and O / GX / 2022). 50 >50 μg·mL⁻¹) and binding activity, which can clearly distinguish the antigenic differences between classic O / Cathay strains and variant strains.

[0012] The present invention also provides an antibody composition for detecting foot-and-mouth disease virus type O, comprising the above-mentioned monoclonal antibodies pO18-40 and / or pO18-43.

[0013] The present invention further provides the application of the above-mentioned monoclonal antibodies pO18-40, pO18-43 or antibody compositions thereof in the preparation of O-type foot-and-mouth disease virus detection reagents or detection kits.

[0014] Preferably, the detection reagent or kit is used to distinguish between classic foot-and-mouth disease virus strains of the O / Cathay lineage and variant foot-and-mouth disease virus strains of the O / Cathay lineage.

[0015] Preferably, the detection reagent or kit detects type O foot-and-mouth disease virus by recognizing amino acid position 149 of the VP1 GH loop region of the foot-and-mouth disease virus VP1 protein.

[0016] The present invention also provides the application of the above-mentioned monoclonal antibodies pO18-40 and / or pO18-43 in screening or optimizing the design of broad-spectrum vaccine strains of type O foot-and-mouth disease virus, and in the analysis of key residues of antigenic structural variations in O / Cathay lineage foot-and-mouth disease strains.

[0017] The monoclonal antibodies pO18-40 and pO18-43 of this invention are whole-porcine monoclonal antibodies. The preparation process is as follows: two IgG strains were screened from the O-type FMDV specific antibody library. The gene sequences of their heavy chain and light chain variable regions were codon-optimized and a signal peptide was added. The heavy chain synthesis gene was inserted into the porcine IgG1 heavy chain constant region gene expression vector, and the light chain synthesis gene was inserted into the porcine κ light chain constant region gene expression vector. The recombinant expression plasmid was transformed into E. coli, amplified, extracted and purified, transfected into CHO-S cells for expression, and purified by affinity chromatography to obtain pure porcine monoclonal antibodies pO18-40 and pO18-43.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The monoclonal antibodies pO18-40 and pO18-43 of the present invention are whole-porcine monoclonal antibodies from pigs susceptible to foot-and-mouth disease. They have higher homology and application compatibility with the natural host and can more realistically simulate the immune response characteristics of the natural host, providing a tool that is closer to the actual infection state for the study of foot-and-mouth disease virus.

[0019] 2. The antibody of this invention has good neutralizing activity against the classical O / Cathay strain O / HN / CHA / 93, but no neutralizing effect on the O / Cathay topological variant strains (O / SCGH / CHA / 2016, O / 18074, O / GD / 2021, O / GX / 2022). It can clearly distinguish the antigenic differences between the classical strain and the variant strain, providing a specific tool for the identification of O-type foot-and-mouth disease virus strains.

[0020] 3. This invention clarifies that both antibodies recognize the GH loop region of the FMDV VP1 protein, and that amino acid position 149 of VP1 in this region is a key site for antibody recognition. It is also a key residue for antigenic variation in the O / Cathay strain and a key site for vaccine immunoprotection. This invention is the first to elucidate the molecular basis of antigenic structural variation in the O / Cathay lineage strains, providing important antigenic information and theoretical basis for the screening and optimization design of broad-spectrum O-type vaccine strains.

[0021] 4. The monoclonal antibody of this invention exhibits good binding ability to the O-type PanAsia lineage strain (O / Xizang / 99), the Mya98 lineage strain (O / GSLX / 2010), and the classic Cathay lineage strain (O / HN / CHA / 93), but lacks binding activity to the Cathay lineage variant strain (O / GX / 2022). Based on its clear and stable antigen recognition difference, it can be used to establish a highly sensitive and specific ELISA detection method, which can not only achieve specific detection of O-type foot-and-mouth disease virus, but also be used to evaluate the neutralizing antibody response titer after FMDV vaccine immunization, thus possessing both diagnostic and immune monitoring application value. The detection reagents / kits constructed based on this antibody are highly sensitive and specific, enabling the differential diagnosis of different FMDV virus antigens.

[0022] 5. The monoclonal antibody preparation method of the present invention is simple, can realize in vitro recombinant expression, has stable yield and high purity, and is suitable for large-scale preparation, laying the foundation for its industrial application. At the same time, the antibody also provides new technical support for basic research on foot-and-mouth disease virus, vaccine development and prevention and control technology upgrade. Attached Figure Description

[0023] Figure 1 The image shows the results of SDS-PAGE electrophoresis to verify the expression of purified porcine monoclonal antibodies pO18-40 and pO18-43. Figure 2 To verify the reactivity of porcine antibodies pO18-40 and pO18-43 with type O FMDV using an indirect immunofluorescence assay; Figure 3 To verify the binding ability of porcine antibodies pO18-40 and pO18-43 to type O FMDV via indirect ELISA, A is the binding curve with O / Xizang / 99, B is the binding curve with O / GSLX / 2010, and C is the binding curve with O / GX / 2022. Figure 4 Alignment of the GH ring amino acid sequence of VP1, a representative strain of type O FMDV; Figure 5 To identify the key sites for porcine monoclonal antibodies to recognize FMDV, where A represents the plaque phenotype of the point mutant strain, B represents the neutralizing efficacy of pO18-40 against the point mutant strain, and C represents the neutralizing efficacy of pO18-43 against the point mutant strain. Figure 6 The neutralizing titer of porcine immune serum against O / 18074 and point mutant strains was determined in the cross-neutralization test. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental materials used in the embodiments of this invention are all conventional biochemical reagents that can be purchased commercially; the experimental methods used are all conventional methods that can be performed with reference to existing technologies or reagent instructions.

[0026] Experimental materials: CHO-S cells, BHK-21 cells, BSR-T7 cells, and FMDV strains (O / Xizang / 99, O / GSLX / 2010, O / HN / CHA / 93, O / SCGH / CHA / 2016, O / 18074, O / GD / 2021, O / GX / 2022, A / AF72, A / WH / CHA / 09, A / GDMM / 2013) were all preserved by the National Foot-and-Mouth Disease Reference Laboratory; the porcine IgGH chain gene vector pVH-pcDNA3.4, the porcine IgGκ chain gene vector pVK-pcDNA3.4, and the porcine IgGλ chain gene vector pVL-pcDNA3.4 were constructed and preserved by the Host Antiviral Infection and Immunobiology Team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0027] The ExpiFectamine™ CHO transfection kit was purchased from Invitrogen. The HiScipt II One Step RT-PCR Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. Mouse anti-pig IgG-FITC antibody was purchased from Mybiosource. Horseradish peroxidase (HRP)-labeled mouse anti-pig tag antibody was purchased from Nanjing Genscript Biotech Co., Ltd. Endotoxin-free plasmid extraction kit was purchased from Tiangen Biotech Co., Ltd.

[0028] Example 1: Preparation of porcine monoclonal antibody specific to foot-and-mouth disease virus type O Two common clone antibodies were removed from the previously established O / 18074 and A / AF72 specific porcine antibody libraries. Two IgG antibodies were screened from the O-type FMDV specific antibody library and named pO18-40 and pO18-43. The heavy and light chain variable region gene sequences of the two antibodies were codon optimized by GenScript Incorporation (www.genscript.com) using CHO-S cells (using Cricetulus griseus). A signal peptide was added to the front end, referring to the literature [Wei Delong. Development of PRRSV-specific monoclonal antibodies based on single B cell antibody technology [D]. Chinese Academy of Agricultural Sciences, 2021. DOI:10.27630 / d.cnki.gznky.2020.000282.]. The heavy chain synthesis gene was inserted into the pCH-pcDNA 3.4 expression vector containing the porcine IgG1 heavy chain constant region gene with tandem HA and His tags at the C-terminus via Not I and BbvCI restriction sites; the porcine κ chain synthesis gene was inserted into the pCK-pcDNA 3.4 expression vector containing the porcine κ light chain constant region gene via Not I and BstBI restriction sites (refer to the literature [Li K, Zhu G, Zhou S, et al. Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus[J].J GenVirol. 2021;102(7):10.1099 / jgv.0.001608]). Recombinant expression plasmids of the antibody heavy and light chains were transformed into E. coli JM109 competent cells for amplification. Single positive colonies were picked and multiplied overnight in Amp-resistant LB medium. The plasmids were extracted and purified using the EndoFreemaxi Plasmid Kit (Tiangen). The heavy / light chain plasmids were transfected into CHO-S cells at a 2:3 mass ratio according to the ExpiFectamine™ CHO Transfection Kit instructions. After 8-9 days of culture, the cell supernatant was collected and purified by affinity chromatography using an AKTA protein purifier. The antibody expression effect was verified by SDS-PAGE.

[0029] The antibody plasmid was expressed in CHO-S cells, purified by affinity chromatography, and the expressed antibody was verified by SDS-PAGE. The results showed that porcine IgG molecules were successfully expressed, with a heavy chain molecular weight of approximately 50 kDa and a light chain molecular weight of approximately 25 kDa. Figure 1The amino acid sequences of the variable region are shown in Table 1 below.

[0030] Example 2: Indirect Immunofluorescence Assay (IFA) for Antibody Reactivity The O / HN / CHA / 93 virus solution was seeded into BHK-21 cells in 24-well cell culture plates at an MOI of 1, with a blank control included. After incubation at 37 ℃ for 3-4 h, pre-cooled fixative (methanol:acetone = 1:1) was added and the cells were fixed at room temperature for 30 min. Indirect immunofluorescence detection was then performed using standard methods. The primary antibody was the expressed antibody (5 µg / mL), and the secondary antibody was FITC-labeled rabbit anti-pig IgG.

[0031] IFA assay results showed that all three antibodies could bind to BHK-21 cells infected with O / HN / CHA / 93 virus, exhibiting specific green fluorescence. Figure 2 The results showed that pO18-40 and pO18-43 could specifically recognize the O / HN / CHA / 93 strain and were reactive to type O FMDV.

[0032] Example 3: Indirect ELISA detection of antibody reactivity Inactivated antigens O / Xizang / 99, O / GSLX / 2010, and O / GX / 2022 were added to ELISA plates at a dose of 100 ng / well and coated overnight at 4°C. The test antibody was diluted to 10 µg / mL and added to the ELISA plates in 10 serial dilutions (2-fold). PBS was used as a negative control. The plates were incubated at 37°C for 1 h. 100 µL of rabbit anti-pig IgG-HRP antibody (1:5000 dilution) was added to each well and incubated at 37°C for 30 min. TMB substrate was added and the plates were incubated at 37°C for 15 min. After stopping the reaction, the OD was measured using an ELISA reader. 450 nm Absorbance at wavelength. When the antibody concentration is diluted to 5 µg / mL, a P / N ratio ≥ 2.1 is considered positive (P is the OD value of two replicate wells for each dilution of the antibody). 450nm The average value, N is the OD of all wells in the PBS control group. 450nm (average of values).

[0033] Indirect ELISA results showed that when the antibody concentration was diluted to 5 µg / mL or higher, under the same antibody concentration conditions, the OD values ​​of pO18-40 and pO18-43 were significantly different from those of O / Xizang / 99 and O / GSLX / 2010. 450nm The values ​​are all higher than the Cut Off value ( Figure 3 AB), and OD of O / GX / 2022 450nm The values ​​are all lower than the Cut Off value ( Figure 3C), CutOff value, also known as CO (positive cutoff value), CO = 2.1 × N (N is the negative control OD). 450nm (Value). This indicates that pO18-40 and pO18-43 can recognize some type O strains (O / Xizang / 99 and O / GSLX / 2010), but cannot recognize the O / Cathay variant strain O / GX / 2022, and are type O FMDV reactive antibodies.

[0034] Example 4: Neutralization Assay to Detect Antibody Neutralization Activity The antibody to be tested was serially diluted twofold in 8 wells of a 96-well plate; 50 µL of viral diluent (titer of 200 TCID50·0.1 mL) was added. -1 Simultaneously, cell controls and virus return controls were set up. After incubation at 37°C for 1 h, 100 µL LBHK-21 cell suspension (density 1×10⁻⁶) was added to each well. 6 mL -1 After 48-72 hours of incubation, CPE was observed. The neutralizing titer of the antibody was calculated using the Reed-Muench method, and the neutralizing power of the antibody (IC50 = antibody concentration / reciprocal of neutralizing titer) was also calculated. IC50 ≤ 50 µg·mL -1 This can be identified as a neutralizing antibody.

[0035] The neutralizing efficacy of the three antibodies was determined using a multi-serotype virus neutralization assay. The results showed that pO18-40 and pO18-43 exhibited good neutralizing activity against the O-type strains O / Xizang / 99, O / GSLX / 2010, and O / HN / CHA / 93, but showed no neutralizing activity against the O / Cathay variant strains from 2016 to 2022, nor against the three A-type strains (A / AF72, A / WH / CHA / 09, and A / GDMM / 2013) (Table 2). These results indicate that these two antibodies are specific neutralizing antibodies against type O FMDV.

[0036] Example 5: Antibody Escape Screening for Antigen Mutation Sites O / HN / CHA / 93 was serially diluted 10-fold to 10. -6 Take 50 µL of virus at each dilution into a 96-well plate and add an equal volume of 50 µg·mL⁻¹. -1 Neutralizing antibodies were added, and antibody-free virus control wells were set up; 100 µL of virus at a concentration of 10⁵ cells / ml was added to each well. -1BHK-21 cells were mixed and incubated at 37°C for 2-3 days. The maximum dilution of virus was taken from 8 wells and passaged continuously in 24-well plates at multiples of the antibody concentration. Viral RNA was extracted from the escaped strains of the third generation, and the FMDV structural protein gene was amplified. The PCR products were sent to a company for sequencing. By comparing the amino acid differences in the structural proteins between the escaped strains and the parental strains, the key amino acids for the neutralizing antibody to recognize viral antigenic epitopes can be identified.

[0037] By screening and comparing the structural protein amino acid differences between neutralizing antibody-escape mutants and parental strains, the key amino acids for antibody recognition were identified. The results (Table 3) showed that the key amino acid for antibody recognition in pO18-40 and pO18-43 was VP1 at position 149 (Q), i.e., residue RGD+2. Comparison with the VP1 GH ring amino acid sequence of the representative O-type FMDV strain revealed that, compared to the Cathay topological classical strain, the 149th amino acid in the mutant strain changed from Q to H (…). Figure 4 This indicates that amino acid position 149 of VP1 is a key site for antibody recognition, and also a key amino acid for antigenic variation in the O / Cathay strain.

[0038] Example 6: Cross-neutralization assay of point-mutated strains to identify key amino acids for antibody recognition Using O / HN / CHA / 93 as the backbone site mutation plasmid ( OHN- VP1 Q149H), with O / 18074 as the backbone, a point mutant plasmid was synthesized ( O18- VP1 H149Q). The cells were digested with Not I enzyme and incubated in a 37°C water bath for 2 h. The linearized DNA product was then recovered using a DNA fragment recovery kit. BSR / T7 cells were cultured in six-well plates. When the cells reached 70%–80% cell growth, the previously linearized DNA was transfected into a monolayer using liposomes and incubated at 37°C. Cytopathic effects were observed every 12 h, and samples showing cytopathic effects were collected and designated as generation 0 single-point mutant virus. Generation 0 virus was repeatedly freeze-thawed three times and then passaged onto BHK-21 cells. Cytopathic effects were observed, and cells reaching 95% or higher were collected and designated as generation 1. Subsequent passages were performed on BHK-21 cells at 10%–20% inoculum until generation 5. The generation 5 single-point mutant virus was serially diluted 10-fold to 10⁻⁶ cells. -6250 µL of the virus solution at its maximum dilution was placed in a six-well plate confluent with BHK-21 cells and incubated at 37°C for 1 hour, agitated every 10 minutes. Then, 2 mL of culture medium (2X MEM and 1.2% astragalus gum in a 1:1 ratio) was added to each well and incubated at 37°C for 48 hours. Ice-cold fixative (50% acetone + 50% methanol) was added and the plate was fixed at -20°C for 30 minutes. Crystal violet was then added for staining, and the staining was performed at room temperature for 30 minutes. The staining solution was discarded and washed away to observe the viral plaques. The TCID of the virus was then measured on BHK-21 cells. 50 The neutralizing efficacy of porcine monoclonal antibodies against successfully rescued single-point mutant strains was determined through a neutralization assay.

[0039] To further verify the escape of key residues from the neutralizing effects of pO18-40 and pO18-43 in the Cathay topological variant strain, and to rescue the VP1 mutant at position 149, a viral plaque assay showed successful rescue. OHN- VP1 Q149H and O18- VP1H149Q single-point mutant strain ( Figure 5 A), and their neutralizing efficacy was evaluated using pO18-40 and pO18-43. The neutralization assay results showed that the Q149H mutant strain with the O / HN / CHA / 93 backbone completely escaped the antibody-mediated virus neutralization of the two strains (A). Figure 5 B); and the neutralizing efficacy of the two antibodies against the O18-VP1H149Q strain was significantly enhanced ( Figure 5 C). These results indicate that amino acid position 149 (RGD+2) of VP1 is a key residue for antigenic variation in the O / Cathay strain.

[0040] Example 7: Cross-neutralization assay of point-mutated strains to identify key amino acids of antigenic variation in the O / Cathay strain Porcine serum immunized with O / HN / CHA / 93 inactivated antigen was inactivated in a water bath at 56°C for 30 min. The serum was then serially diluted twofold in eight wells of a 96-well plate; 50 µL of virus diluent (titer 200 TCID50·0.1 mL) was added to each well. -1 Simultaneously, cell controls and virus return controls were set up. After incubation at 37°C for 1 h, 100 µL of BHK-21 cell suspension (density 1×10⁻⁶) was added to each well. 6 mL -1 After 48 h to 72 h of culture, CPE was observed, and the neutralizing titer of serum was calculated using the Reed-Muench method.

[0041] The cross-neutralization assay was used to determine the effectiveness of immune serum against O / 18074 and point mutant strains. O18-The neutralizing titer of VP1 H149Q was determined, and the results showed that, compared with O / 18074, the neutralizing titer of immune serum against the single-point mutant strain was significantly increased. Figure 6 This result indicates that amino acid position 149 (RGD+2) of VP1 is a key residue for antigenic variation in the O / Cathay strain and a critical site for vaccine-mediated immune protection.

Claims

1. A porcine monoclonal antibody pO18-40 that neutralizes type O foot-and-mouth disease virus, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody pO18-40 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. A porcine monoclonal antibody pO18-43 that neutralizes type O foot-and-mouth disease virus, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody pO18-43 is shown in SEQ ID No. 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

4.

3. An antibody composition for detecting foot-and-mouth disease virus type O, characterized in that, It comprises the monoclonal antibody pO18-40 of claim 1 and / or the monoclonal antibody pO18-43 of claim 2.

4. The use of the monoclonal antibody pO18-40 of claim 1, the monoclonal antibody pO18-43 of claim 2, or the antibody composition of claim 3 in the preparation of a type O foot-and-mouth disease virus detection reagent or kit.

5. The application according to claim 4, characterized in that, The detection reagents or kits are used to distinguish between classic foot-and-mouth disease virus strains of the O / Cathay lineage and variant foot-and-mouth disease virus strains of the O / Cathay lineage.

6. The application according to claim 4, characterized in that, The detection reagent or kit detects type O foot-and-mouth disease virus by recognizing amino acid position 149 of the VP1 ring region of the VP1 protein of foot-and-mouth disease virus.

7. The use of the monoclonal antibody pO18-40 of claim 1 or the monoclonal antibody pO18-43 of claim 2 in screening or optimizing the design of broad-spectrum vaccine strains for type O foot-and-mouth disease virus.

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