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

Abstract

The invention discloses a foot-and-mouth disease virus type O specific neutralizing swine monoclonal antibody and application thereof, and belongs to the technical field of biology. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2. The invention also provides application of the monoclonal antibody in preparation of a reagent or a kit for detecting the O-type foot-and-mouth disease virus and a medicine for preventing O-type foot-and-mouth disease virus infection. The antibody is a whole swine source, is good in safety, can specifically neutralize O-type FMDV PanAsia, Mya98 and Cathay classical pedigree strains, identifies key antigen sites as 73rd and 134 residues of VP3, can be used for O-type FMDV differential diagnosis and broad-spectrum vaccine design, and has an important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a porcine monoclonal antibody that is specific to type O foot-and-mouth disease virus and its application. Background Technology

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease caused by the foot-and-mouth disease virus (FMDV), primarily affecting cloven-hoofed animals such as pigs, cattle, and sheep. It severely restricts international trade in animals and animal products, causing enormous economic losses to the global livestock industry. FMDV belongs to the Picornaviridae family and, based on antigenic differences, can be classified into seven serotypes: A, O, C, Asia1, SAT1, SAT2, and SAT3. There is no cross-protection between different serotypes, and multiple topotypes and lineages exist within the same serotype.

[0003] In my country, type O FMDV is the most serious serotype threatening livestock farming. In recent years, it has shown a mixed prevalence of three topotypes and four lineages: Cathay, ME-SA / PanAsia, ME-SA / Ind-2001, and SEA / Mya98. Among them, the O / Cathay topotype virus is highly swine-loving and has formed a viral group with unique genetic characteristics over more than fifty years of prevalence. In recent years, the evolutionary rate of this topotype virus has accelerated significantly, its genetic diversity has increased dramatically, and significant antigenic variations have emerged.

[0004] Currently, vaccination is one of the main methods for controlling foot-and-mouth disease (FMD). However, the antigenic matching between existing type O vaccine strains and prevalent O / Cathay topological variants continues to decline, significantly reducing the effectiveness of vaccines. This suggests that important structural variations may have occurred at the antigenic sites of the O / Cathay topological variants. Therefore, it is urgent to discover type O FMDV-specific neutralizing antibodies, especially those derived from the natural host, pigs. By analyzing their binding mechanism with the virus and the key antigenic sites they recognize, we can elucidate the molecular basis of O / Cathay topological virus antigenic variation, providing crucial antigenic structural information and theoretical support for the optimized design of type O broad-spectrum FMDV vaccines. This will also provide a material basis for establishing specific type O FMDV detection methods. Summary of the Invention

[0005] The purpose of this invention is to provide a porcine monoclonal antibody pO18-29 that is specific to type O foot-and-mouth disease virus and its application.

[0006] This invention is achieved through the following technical solution: This invention provides a porcine monoclonal antibody pO18-29 that is specific to neutralize type O foot-and-mouth disease virus. The monoclonal antibody pO18-29 includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region 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.

[0007] Heavy chain variable region: EDKVVESGGGLVQPGGSLRLSCVGSVWDFPNYPFTWVRQAPGKGLEWLASIGCGVNSGATHYADSVKGRFTISRDNSVTTASLQMDKLRTEDTARYYCTSGRRKNVGYCYRDFVGYNPLNVWGPGVEVVVTS (SEQ ID No. 1).

[0008] Light chain variable region: QTVIQEPAMSVSPGGTVTLTCALSSGSVTSDDYPGWYQQTPGQPPRQLIYGTDNRLTGVSRRFSGVISENKATLTISGAQAEDEADYFCDLYKSNMKIFGGGTHLTVL (SEQ ID No. 2).

[0009] The present invention encodes a DNA fragment of the foot-and-mouth disease virus O-specific neutralizing porcine monoclonal antibody pO18-29, the DNA fragment encoding the heavy chain variable region is shown in SEQ ID NO.3; and the DNA fragment encoding the light chain variable region is shown in SEQ ID NO.4.

[0010] Heavy chain variable region DNA fragment: GAGGACAAAGTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGTTTTGGGACTTCCCAAACTACCCTTTCACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTGGCAAGTATTGGTTGTGGTGTTAATAGTGGTGCCACCCACTACGCAGACTCTGTGAA GGGCCGATTCACCATCTCCAGAGACAATTCCGTAACCACGGCCTCTCTGCAAATGGACAAACTGAGAACCGAAGACACGGCCCGCTATTATTGTACTTCAGGTCGGCGGAAAAACGTTGGGTATTGCTACCGCGATTTCGTTGGTTATAATCCGTTGAATGTCTGGGGCCCAGGCGTTGAAGTCGTCGTGACCTCAG (SEQ ID NO.3).

[0011] Light chain variable region DNA fragment: CAGACTGTGATCCAGGAGCCGGCGATGTCAGTGTCTCCTGGAGGGACCGTCACACTCACCTGTGCCTTGAGCTCTGGGTCAGTCACTAGTGATGACTACCCTGGCTGGTACCAGCAGACACCAGGCCAGCCTCCCCGACAACTAATCTACGGCACAGACAACCG CCTGACTGGGGTCTCCAGGCGCTTCTCTGGAGTCATCTCTGAGAACAAAGCCACCCTCACCATCTCGGGGGCCCAGGCTGAGGACGAGGCCGACTATTTTTGTGATCTTTATAAAAGTAATATGAAGATTTTCGGCGGTGGGACCCATCTGACCGTTCTCA (SEQ IDNO.4).

[0012] A vector comprising the DNA fragment. A host cell comprising the vector.

[0013] The present invention also provides applications of the above-mentioned monoclonal antibody pO18-29, specifically including its application in the preparation of a test reagent or kit for type O foot-and-mouth disease virus, and its application in the preparation of a drug for preventing type O foot-and-mouth disease virus infection.

[0014] The monoclonal antibody pO18-29 specifically recognizes PanAsia lineage (e.g., O / Xizang / 99), Mya98 lineage (e.g., O / GSLX / 2010), and classic Cathay lineage strains (e.g., O / HN / CHA / 93) of type O FMDV, but does not recognize Cathay lineage variants (e.g., O / GX / 2022, O / 18074, O / GD / 2021). Therefore, based on this characteristic, detection reagents or kits, such as ELISA kits, can be prepared to distinguish between classic and variant strains of type O FMDV Cathay topology. Simultaneously, this antibody exhibits good neutralizing activity against the aforementioned identifiable type O FMDV strains and can be used to prepare drugs for the prevention of corresponding type O FMDV infections.

[0015] Further research showed that the key antigenic sites for the monoclonal antibody pO18-29 to recognize type O FMDV are residues 73 (V) and 134 (K) of the capsid protein VP3. These two sites are key amino acids that cause neutralization and escape of Cathay topological strains. This discovery can provide a clear antigenic structural basis for the molecular design of type O broad-spectrum foot-and-mouth disease vaccines.

[0016] The monoclonal antibody pO18-29 provided by this invention is derived from the natural host of FMDV, the pig. It is a whole-porcine monoclonal antibody with good species homology and application safety. This antibody exhibits specific neutralizing activity against type O FMDV. Its key binding target has been clearly identified at residues 73 and 134 of the capsid protein VP3, providing a clear antigenic structural basis for the molecular design of broad-spectrum type O vaccine strains, thus enabling its use in the prevention of FMDV infection. pO18-29 shows good binding ability to the type O PanAsia lineage (O / Xizang / 99), Mya98 lineage (O / GSLX / 2010), and the classic Cathay lineage strain (O / HN / CHA / 93), but has no binding activity against the variant strain of the Cathay lineage (O / GX / 2022). Based on its clear antigen recognition differences, this antibody can be used to establish a highly sensitive and specific ELISA detection method for the differential diagnosis of FMDV viral antigens.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The monoclonal antibody pO18-29 provided by this invention is a whole-pig monoclonal antibody. Its gene is derived from the natural host of FMDV, the pig. It has good species homology and application safety, avoiding problems such as immune rejection that may be caused by heterologous antibodies, and is more suitable for use in pigs.

[0018] 2. The monoclonal antibody pO18-29 has specific neutralizing activity against type O FMDV and can specifically neutralize classic strains of the PanAsia, Mya98 and Cathay lineages, providing a new biological agent option for the prevention of infection by these strains.

[0019] 3. This invention clarifies that the key antigenic sites for the monoclonal antibody pO18-29 to recognize type O FMDV are residues 73 and 134 of VP3, revealing the molecular basis for the neutralization and escape of Cathay topological variant strains. It provides key antigenic structural information and theoretical support for the optimized design of type O broad-spectrum foot-and-mouth disease vaccines, and is of great significance for promoting the development of foot-and-mouth disease prevention and control technology.

[0020] 4. The monoclonal antibody pO18-29 has a clear distinguishing effect on different lineages / strains of type O FMDV. It can bind to classic strains but not to variant strains. Based on this characteristic, a detection method with good sensitivity and high specificity can be established to achieve differential diagnosis of type O FMDV antigens, providing a powerful tool for the epidemiological monitoring and prevention and control of foot-and-mouth disease. Attached Figure Description

[0021] Figure 1 The expression and purification of the porcine monoclonal antibody pO18-29 were verified by SDS-PAGE electrophoresis. Figure 2 To verify the reactivity of porcine antibody pO18-29 with type O FMDV using an indirect immunofluorescence assay; Figure 3 To verify the binding ability of porcine antibody pO18-29 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 To resolve the structure of the porcine monoclonal antibody-FMDV complex using cryo-electron microscopy, A is an extended view of the interaction interface between pO18-29 scFv and the O / HN / CHA / 93 capsid, and B is an extended view of the interaction interface between pO18-29 scFv and the O / 18074 capsid. Figure 5 Amino acid sequence alignment of VP3, a representative strain of type O FMDV; Figure 6 To identify key sites for FMDV recognition by porcine monoclonal antibodies in a cross-neutralization assay, A represents the neutralizing efficacy of pO18-29 against O / HN / CHA / 93 and its mutants, and B represents the neutralizing efficacy of pO18-29 against O / 18074 and its mutants. Detailed Implementation

[0022] The present invention will be further described below through specific embodiments.

[0023] Key materials used in this invention: BHK-21 cells, CHO-S 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 and the porcine IgGλ chain gene vector pVL-pcDNA3.4 were constructed and preserved by the Host Antiviral Infection and Immunobiology Team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The one-step RT-PCR amplification kit (HiScipt II One Step RT-PCR Kit) was purchased from Nanjing Novizan Biotechnology Co., Ltd. E. coli JM109 competent cell line was purchased from Taraka Biotechnology Co., Ltd. Mouse anti-pig IgG-FITC antibody was purchased from Mybiosource. Endotoxin-free plasmid extraction kit was purchased from Tiangen Biotech Co., Ltd.

[0024] Example 1: Preparation of porcine monoclonal antibody specific to foot-and-mouth disease virus type O After removing 216 common clone antibodies from the previously established O / 18074 and A / AF72 specific porcine antibody libraries, one IgG antibody, named pO18-29, was screened from the O-type FMDV specific antibody library. The heavy and light chain variable region gene sequences were optimized using the CHO-S cell codon by GenScript, and a secretion signal peptide was added to the front end, referring to the literature [Wei Delong. Development of PRRSV-specific monoclonal antibody 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 IgG λ chain synthesis gene was inserted into the pVL-pcDNA3.4 vector containing the porcine IgG λ chain gene via Not I and Ale I restriction sites, referring 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 EndoFree maxi 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 culturing for 7-8 days, the cell supernatant was collected and purified by affinity chromatography using an AKTA protein purifier. The antibody expression effect was verified by SDS-PAGE.

[0025] The antibody plasmid was expressed in CHO-S cells, purified by affinity chromatography, and the expression of the antibody was verified by SDS-PAGE. The results showed that porcine IgG molecules pO18-29 were successfully expressed, with a heavy chain molecular weight of approximately 50 kDa and a light chain molecular weight of approximately 25 kDa. Figure 1 ).

[0026] Example 2: Indirect Immunofluorescence Assay (IFA) for Detecting 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 4 ℃ for 30 min. Indirect immunofluorescence detection was then performed using standard methods. The primary antibody was a porcine monoclonal antibody (working concentration 5 µg / mL), and the secondary antibody was FITC-labeled rabbit anti-porcine IgG.

[0027] IFA assay results showed that pO18-29 could bind to BHK-21 cells infected with O / HN / CHA / 93 virus, exhibiting specific green fluorescence. Figure 2 The results showed that pO18-29 can specifically recognize the O / HN / CHA / 93 strain and is reactive to type O FMDV.

[0028] 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 of the values, where N is the OD of all wells in the PBS control group. 450nm (average of values).

[0029] 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 pO18-29, O / Xizang / 99, and O / GSLX / 2010 were significantly different. 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 3 C), Cut-Off value, i.e., CO (positive cut-off value), CO = 2.1 × N (N is the negative control OD). 450nm(Value). This indicates that pO18-29 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 is a type O FMDV reactive antibody.

[0030] Example 4: Neutralization Assay to Detect Antibody Neutralization Activity The antibody to be tested was serially diluted 2-fold in 8 wells of a 96-well plate; 50 µL of viral diluent (titer of 200 TCID) was added. 50 0.1 / mL), with cell control and virus return control set up simultaneously. After incubation at 37℃ for 1 h, 100 µL of BHK-21 cell suspension (density 1×10⁻⁶) was added to each well. 6 mL -1 After culturing for 48-72 hours, cytopathic effects (CPE) were observed. The neutralizing titer of the antibody was calculated using the Reed-Muench method, and the neutralizing potency (IC50) of the antibody was also calculated. 50 = (antibody concentration / reciprocal of neutralizing titer), IC 50 A concentration of ≤50µg / mL is considered a neutralizing antibody.

[0031] The neutralizing efficacy of the antibody was determined by a neutralization assay for type O FMDV. The results showed that pO18-29 could neutralize four type O strains (O / Xizang / 99, O / GSLX / 2010, O / HN / CHA / 93, O / SCGH / CHA / 2016), but had no neutralizing activity against three type O Cathay topological variants (O / 18074, O / GD / 2021, O / GX / 2022) produced after 2018, and also had no neutralizing activity against three representative type A strains (A / AF72, A / WH / CHA / 09, A / GDMM / 2013) (Table 2). These results indicate that pO18-29 is a type O FMDV-specific neutralizing antibody.

[0032] Example 5: Western blot analysis of antigenic epitopes recognized by antibodies The purified 146S antigen of O / HN / CHA / 93 was subjected to SDS-PAGE, and the protein was then transferred to a PVDF membrane for Western blotting verification. The primary antibody was a porcine monoclonal antibody (working concentration 2 µg / mL), incubated at room temperature for 2 h; the secondary antibody was rabbit anti-porcine IgG-HRP antibody (1:5000 dilution), incubated at room temperature for 1 h, and then exposed using an exposure meter for recording.

[0033] Western blot assays were performed using the O / HN / CHA / 93 inactivated antigen to identify the epitope type recognized by the neutralizing antibody. The results showed that pO18-29 did not bind to denatured type O and FMDV antigens, indicating that pO18-29 recognizes the conformational epitope of FMDV.

[0034] Example 6: Cryo-electron microscopy analysis of the antigen-antibody complex structure Plasmids containing the pO18-29 single-stranded variable region fragment (scFv) were designed and synthesized. Antibodies were expressed in CHO-S cells, and pOA-2-scFv was purified by affinity chromatography and molecular sieving. O / HN / CHA / 93 and O / 18074 viruses were inactivated, and the 146S antigen was purified by sucrose gradient and ultracentrifugation. The prepared pO18-29-scFv fragment was mixed with O / HN / CHA / 93 and O / 18074 146S antigens at a ratio of 120:1 (molar ratio), and incubated on ice for 30 min. Then, 3 μL of the FMDV-OHN-pO18-29 and FMDV-O18-pO18-29 complexes were added to copper mesh embedded with carbon film (400 mesh), and the copper mesh was flash-frozen in liquid ethane at 90% humidity for 3 seconds, and then transferred to a liquid nitrogen tank for storage. Data were collected using a 300 kV cryomicroscope equipped with a K2 detector, and the complex structure was analyzed based on electron density characteristics.

[0035] Cryo-electron microscopy results showed that the FMDV structural proteins VP1 to VP4 were blue, green, red, and yellow, respectively; the heavy chain variable region (VH) and light chain variable region (VL) of pO18-29 were light blue and purple, respectively; and the putative hydrogen bonds and salt bridges were marked with black or red dashed lines. Figure 4The FMDV-OHN-pO18-29 complex forms 17 hydrogen bonds and 5 salt bridge interfaces through interacting residues (Table 3). The FMDV-O18-pO18-29 complex forms 25 hydrogen bonds and 7 salt bridge interfaces through interacting residues (Table 4). While the two complexes are structurally similar, significant differences exist at key interaction interfaces. First, in the FMDV-OHN–pO18-29 complex, a strong hydrophobic interaction exists between the VP3 β-C sheet of O / HN / CHA / 93 and the HCDR3 of pO18-29, with its core composed of the V73 residue of VP3 and the I106 residue of HCDR3. However, in the FMDV-O18–pO18-29 complex, this hydrophobic core completely disappears due to the substitution of VP3V73K, resulting in the absence of the relevant hydrophobic contact. Secondly, in O / HN / CHA / 93, residue 134 of VP3 forms two salt bridges and one hydrogen bond with pO18-29; conversely, in O / 18074, the corresponding residues cannot form hydrogen bonds or salt bridges with the antibody, indicating a significant weakening of interfacial electrochemical interactions. These results suggest that the pO18-29-FMDV binding region is the structural basis for the neutralization escape of the Cathay topological variant, with key amino acid residues located in the EF loop of the FMDV VP3 protein, revealing the structural basis for the neutralization escape of the Cathay topological variant.

[0036] Sequence alignment of the O-type FMDV representative strain further confirmed that, between the classic O / Cathay strain and the variant strain, mutations occurred at residues 73 and K134 of VP3. At residue 73, the basic amino acid K was replaced by the nonpolar hydrophobic amino acid V, and at residue 134, the neutral amino acid was replaced by the positively charged K. Figure 5 These amino acid changes may lead to significant alterations in the local conformation and interfacial chemical environment of the viral capsid surface, thereby disrupting the key hydrophobic and electrochemical interactions required for antibody recognition, ultimately allowing the O / Cathay mutant to escape the neutralizing effect of the pO18-29 antibody.

[0037] Example 7: Cross-neutralization assay of point mutant strains to identify key amino acids for antibody recognition Three point mutant plasmids were synthesized using O / HN / CHA / 93 as the backbone. OHN- VP3 V73K OHN- VP3 K134T, OHN- VP3V73K / K134T, using O / 18074 as the backbone, synthesized 3 point mutant plasmids ( O18- VP3 K73V, O18-VP3 T134K O18- VP3K73V / T134K). The DNA was digested with Not I enzyme and incubated at 37°C 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% confluency, 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 passage 0 single-point mutant virus. The passage 0 virus was repeatedly frozen and thawed three times and then passaged in BHK-21 cells. Cytopathic effects were observed, and cells reaching 95% or higher confluency were collected and designated as passage 1. Subsequent passages were performed in BHK-21 cells at 10%–20% inoculum up to passage 5. The TCID of the virus was measured on BHK-21 cells. 50 The neutralizing efficacy of pO18-29 against successfully rescued single-point mutant strains was determined through a neutralization test.

[0038] To further verify the escape of key residues from pO18-29 neutralization by the Cathay topological mutant strain, the cross-neutralization test results between the point mutant strain and pO18-29 are as follows: Figure 6 As shown, pO18-29 showed no significant difference in neutralizing efficacy against mutant strains with the O / HN / CHA / 93 backbone (including mutations at positions 73, 134, and 73 / 134). However, for mutant strains with the O / 18074 backbone, the neutralizing efficacy was significantly enhanced, especially in double-point mutant strains where both positions 73 and 134 of VP3 were substituted, where the increase in neutralizing efficacy was most pronounced. These results indicate that V73 and K134 of VP3 are key residues affecting the antigenicity changes of O / Cathay mutant strains, and these two sites exhibit a synergistic effect in the neutralization escape of mutant strains. This suggests that residues at positions 73 (V) and 134 (K) of VP3 jointly determine the antigenicity changes of O / Cathay mutant strains and play a synergistic role in neutralization escape.

Claims

1. A porcine monoclonal antibody specifically neutralizing type O foot-and-mouth disease virus, characterized in that: The monoclonal antibody It includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region 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. The monoclonal antibody according to claim 1, characterized in that: The DNA sequence encoding the heavy chain variable region is shown in SEQ ID NO.3, and the DNA sequence encoding the light chain variable region is shown in SEQ ID NO.

4.

3. A carrier, characterized in that: It contains the DNA sequence as described in claim 2.

4. A host cell, characterized in that: It includes the carrier as described in claim 3.

5. The use of the monoclonal antibody according to claim 1 in the preparation of a foot-and-mouth disease virus type O detection reagent or kit.

6. The application according to claim 5, characterized in that, The detection reagent or kit is used to distinguish between classical strains and variant strains of type O foot-and-mouth disease virus Cathay topology.

7. The application according to claim 5, characterized in that, The classical strains of the O-type foot-and-mouth disease virus Cathay topology include O / HN / CHA / 93, and the variant strains include O / GX / 2022, O / 18074, and O / GD / 2021.

8. The use of the monoclonal antibody pO18-29 according to claim 1 in the preparation of a medicament for preventing foot-and-mouth disease virus infection of type O.

9. The application according to claim 8, characterized in that, The type O foot-and-mouth disease virus includes classic strains of the PanAsia, Mya98, and Cathay lineages.

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