Preparation and application of monoclonal antibody of O / Cathay type foot-and-mouth disease virus and variable region sequence of the antibody

Abstract

The application belongs to the technical field of biology, and particularly relates to a mouse-derived O / Cathay type foot-and-mouth disease virus monoclonal antibody and preparation and application thereof. The application obtains a monoclonal antibody 3G10 against the O / Cathay type foot-and-mouth disease virus; the prepared monoclonal antibody 3G10 can specifically react with the O / Cathay type foot-and-mouth disease virus, has the advantages of high specificity, sensitivity, low preparation cost and the like, provides new materials for serotype identification and quantification of the O / Cathay type foot-and-mouth disease virus, and provides new technical support for controlling the spread of the foot-and-mouth disease epidemic; the monoclonal antibody 3G10 provided by the application can be used for recombination and modification by a conventional genetic engineering or protein engineering method, antibody loss caused by long-term freezing of hybridoma cells is avoided, and the antibody is also beneficial to optimization at the genetic and protein levels, so that the specificity and affinity of the antibody are improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation of a mouse-derived O / Cathay type foot-and-mouth disease virus monoclonal antibody 3G10 and its application in the ELISA detection method for O / Cathay type foot-and-mouth disease virus typing and quantification. Background Technology

[0002] Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and is highly pathogenic and contagious in cloven-hoofed animals such as pigs, cattle, and sheep. There are 37 serotypes of FMDV: O, A, Asia1, C, SAT1, SAT2, and SAT. Among them, O, A, and Asia1 are the most widely reported FMDV serotypes to the World Organisation for Animal Health (WOAH). Serotype O includes eleven topotypes, such as ME-SA, SEA, and CATHAY. The global FMDV epidemic is generally becoming more complex, and there is no cross-protection between serotypes conferred by vaccination or previous infection. The ability to mutate easily is an inherent characteristic of RNA viruses, allowing them to evade antibody neutralization. Continuous viral mutations and the emergence of circulating variants make the eradication of FMDV exceptionally challenging. The O-type (CATHAY) foot-and-mouth disease virus circulating in my country in recent years differs significantly from previously prevalent viruses. Existing vaccines are no longer effective against the new CATHAY strain, necessitating the design, development, and mass production of new vaccine strains to improve protective efficacy. However, currently, there is a lack of effective antigen quantification methods for the O / CATHAY strain. After updating vaccine components with the O / CATHAY strain antigens, it is impossible to rapidly quantify the content of the old O-type antigen and O-type (CATHAY) components in the vaccine within a short period. This makes post-production quality control and efficacy evaluation difficult.

[0003] Foot-and-mouth disease virus (FMD) typing refers to determining the specific type of the virus, such as type O, type A, or Asia, through laboratory testing methods. Differentiating between the O / CATHAY serotype and other types helps in identifying external transmission, tracing the source of the outbreak, and developing more targeted prevention and control strategies. Different FMD virus types possess different antigenicities, requiring vaccines and control measures to be designed for specific types. The distribution and variations of viral antigenic epitopes significantly impact vaccine efficacy; typing ensures that the vaccine matches the circulating virus type, avoiding immunization failure due to type mismatch.

[0004] Currently, the main methods for typing foot-and-mouth disease virus (FMDV) include real-time fluorescent RT-PCR, colloidal gold immunochromatography (GICA), and ELISA. While RT-PCR and GICA offer advantages in rapid detection, they suffer from drawbacks such as demanding reaction environment requirements and a certain false-positive rate. ELISA offers a balanced advantage in sensitivity, specificity, quantification, standardization, and automation, reflecting the presence of viral particles and being suitable for large-scale serum sample testing. Coating with universal capture antibodies to capture the FMDV 146S antigen enhances the specific capture of the effective antigen (i.e., the 146S antigen). Monoclonal antibodies recognize only specific antigenic epitopes, accurately identifying and binding trace amounts of antigen, exhibiting high specificity and sensitivity, uniform properties, and reproducible production processes, making them suitable for large-scale production. Using both methods simultaneously to establish a quantitative ELISA detection method for FMDV type O (CATHAY) typing in inactivated FMD vaccines improves the convenience and accuracy of the detection method, providing a reference for antigen content quantification and vaccine efficacy evaluation during vaccine production. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention has screened and obtained a murine monoclonal antibody, 3G10, against foot-and-mouth disease virus (FMDV), which exhibits high specificity, good biological activity, and strong antigen-binding ability. Using monoclonal antibody 3G10 and a universally trapping rabbit antibody, a genotyping and quantitative ELISA method for O / Cathay type FMDV was established. This method enables serotyping diagnosis and precise antigen quantification of O / Cathay type FMDV, providing a reference for post-production quality control and efficacy evaluation of vaccines, and is of significant importance in FMDV prevention and control. Specifically, it includes the following:

[0006] In a first aspect, the present invention provides an O / Cathay type foot-and-mouth disease virus monoclonal antibody, wherein the O / Cathay type foot-and-mouth disease virus monoclonal antibody comprises an antibody heavy chain and an antibody light chain;

[0007] The variable region CDR of the antibody heavy chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3;

[0008] The variable region CDR of the antibody light chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No. 6.

[0009] Preferably, the O / Cathay type foot-and-mouth disease virus monoclonal antibody comprises an antibody heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 7 and an antibody light chain variable region with an amino acid sequence as shown in SEQ ID No. 8.

[0010] Secondly, the present invention provides a nucleic acid molecule that encodes the O / Cathay type foot-and-mouth disease virus monoclonal antibody described in the first aspect above.

[0011] Preferably, the nucleic acid molecule comprises the nucleotide sequences shown in SEQ ID No. 9 and SEQ ID No. 10.

[0012] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect above.

[0013] Fourthly, the present invention provides a recombinant cell comprising the nucleic acid molecule described in the second aspect above, or the expression vector described in the third aspect above.

[0014] Fifthly, the present invention provides the application of the O / Cathay type foot-and-mouth disease virus monoclonal antibody described in the first aspect above in the preparation of products for the genotyping and quantitative detection of O / Cathay type foot-and-mouth disease virus.

[0015] In a sixth aspect, the present invention provides the application of the O / Cathay type foot-and-mouth disease virus monoclonal antibody described in the first aspect above in the quantitative detection of O / Cathay type foot-and-mouth disease virus for non-disease diagnosis purposes.

[0016] In a seventh aspect, the present invention provides a quantitative detection kit for O / Cathay type foot-and-mouth disease virus, the kit comprising the O / Cathay type foot-and-mouth disease virus monoclonal antibody described in the first aspect above.

[0017] Preferably, the kit further includes an enzyme-labeled plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

[0018] Eighthly, the present invention provides a method for preparing a monoclonal antibody against O / Cathay type foot-and-mouth disease virus as described in the first aspect above. The method comprises: purifying and inactivating O / Cathay type foot-and-mouth disease virus using PEG1500; immunizing Balb / c mice with the purified and inactivated O / Cathay type foot-and-mouth disease virus as an antigen; fusing mouse spleen cells with myeloma cells SP2 / 0 to prepare hybridoma cells; verifying the cell supernatant by indirect ELISA and screening for positive clones; after three subcloning processes, injecting the hybridoma cells into mice to prepare ascites; and finally purifying the obtained ascites to obtain a monoclonal antibody 3G10 against O / Cathay type foot-and-mouth disease virus.

[0019] In a ninth aspect, the present invention provides a method for preparing the O / Cathay type foot-and-mouth disease virus monoclonal antibody described in the first aspect above. The method comprises: linking the heavy chain variable region and light chain variable region sequences of the O / Cathay type foot-and-mouth disease virus monoclonal antibody with the heavy chain constant region and light chain constant region sequences of the antibody, respectively, and inserting them into an expression vector to obtain a recombinant expression plasmid of antibody heavy chain and antibody light chain; co-transfecting the obtained recombinant expression plasmid into expression cells and culturing to obtain the O / Cathay type foot-and-mouth disease virus monoclonal antibody.

[0020] The beneficial effects of this invention are as follows: This invention utilizes PEG 1500 to purify and inactivate O / Cathay type foot-and-mouth disease virus, which is then used as an immunogen to immunize mice. Through cell fusion and subcellular screening, a monoclonal antibody 3G10 targeting O / Cathay type foot-and-mouth disease virus was successfully obtained. The prepared monoclonal antibody 3G10 can specifically react with O / Cathay type foot-and-mouth disease virus, exhibiting advantages such as high specificity, sensitivity, and low preparation cost. It provides new materials for serotype identification and quantification of O / Cathay type foot-and-mouth disease virus and also provides new technical support for controlling the spread of foot-and-mouth disease. This invention also provides the sequence of monoclonal antibody 3G10, which can be used for recombination and modification using conventional genetic engineering or protein engineering methods. This avoids antibody loss during long-term cryopreservation of hybridoma cells and also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 Purification results of monoclonal antibody 3G10;

[0023] Figure 2 Western blot (WB) results of monoclonal antibody 3G10 reacting with inactivated O / Cathay type foot-and-mouth disease virus;

[0024] Figure 3 Results of titer assay for the prepared monoclonal antibody 3G10

[0025] Figure 4 Results of monoclonal antibody 3G10 subtype identification;

[0026] Figure 5 Specificity identification results of monoclonal antibody 3G10;

[0027] Figure 6 Method for calculating antigen content in quantitative detection of O / Cathay type foot-and-mouth disease virus;

[0028] Figure 7 Validation results of the accuracy verification of the O / Cathay type foot-and-mouth disease virus typing and quantitative detection method;

[0029] Figure 8 Repeatability verification results of the O / Cathay type foot-and-mouth disease virus typing and quantitative detection method;

[0030] Figure 9 Specificity validation results of the O / Cathay type foot-and-mouth disease virus typing and quantitative detection method. Detailed Implementation

[0031] The present invention will be described in detail below through specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. Any technical solution that can be conceived by those skilled in the art based on the present invention and in combination with common knowledge in the art shall fall within the scope of protection of the present invention. In addition, for those embodiments where specific technical operation steps or conditions are not specified, they shall be performed in accordance with the techniques or conditions described in general literature in the art or in accordance with the product instructions. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.

[0032] Light chains are classified into κ chains or λ chains based on their constant regions. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known to those skilled in the art. In this invention, VH represents the variable region of the heavy chain, and VL represents the variable region of the light chain, which is divided into κ and λ types.

[0033] The materials used in the following examples include: BALB / c mice purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; HRP Conjugation Kit-Lighting- The kits were purchased from Abcam, the protein G column packing material, Freund's complete and incomplete adjuvants were purchased from Sigma, the mouse monoclonal antibody subtype identification kit was purchased from Proteintech, Trizol, the DNA fragment recovery kit and the plasmid extraction kit were OMEGA products, the molecular biology reagents were from Sigma, and other biochemical reagents were domestically produced analytical grade reagents.

[0034] The foot-and-mouth disease viruses involved in the following implementations include: O-GXCX-CHA-2018-S strain (GenBank accession number MH791316), O / BY / 2010 (GenBank accession number JN998085), O / MYA98 (GenBank accession number JN998086), OZK / 93 (commercial vaccine strain, reference: Bai Xingwen. Molecular basis of phenotypic differences of O-type Pan-Asian 1-line foot-and-mouth disease virus in my country [D]. Chinese Academy of Agricultural Sciences, 2012.), AF / 72 (GenBank accession number MT442614), A / GDMM / 2013 (GenBank accession number KF450794), and A / WH / 09 (GenBank accession number JF792355).

[0035] The O / Cathay type foot-and-mouth disease virus described in the following examples is O-GXCX-CHA-2018-S.

[0036] Example 1: Preparation of monoclonal antibody against mouse O / Cathay type foot-and-mouth disease virus

[0037] 1.1 Immunizing mice

[0038] Inactivated O / Cathay type foot-and-mouth disease virus was purified using PEG1500 as the inactivated antigen. The purified O / Cathay type foot-and-mouth disease virus inactivated antigen was diluted to 200 μg / mL with PBS and emulsified with an equal volume of Freund's complete adjuvant. Six 6-week-old female BALB / c mice were subcutaneously injected with 200 μL of the emulsified antigen at multiple sites on their backs. Two booster immunizations were administered on days 15 and 30 after the initial immunization using Freund's incomplete adjuvant, following the same method. Antibody levels were measured one week after the third immunization via tail vein. [The text abruptly ends here, likely due to an incomplete source.] 450nm Mice with a growth rate >2.0 were given a booster immunization three days prior to fusion by intraperitoneal injection of 0.5 mL of unadjuvanted antigen.

[0039] 1.2 Cell Fusion

[0040] Positive serum was prepared from blood collected from BALB / c mice 3 days after booster immunization. Mice were euthanized, and spleens were aseptically harvested after disinfection, washed, and fat removed. The spleens were minced and ground in incomplete RPMI-1640, and spleen cells were collected. Spleen cells were mixed with Sp2 / 0 cells at a ratio of 1:5 to 1:10, centrifuged, and then fused with PEG2000. The fused cells were then diluted with incomplete RPMI-1640. The fused cells were resuspended in HAT selective medium, seeded in 96-well plates, and cultured at 37°C with 5% CO2.

[0041] 1.3 Screening and subcloning of positive hybridoma cells

[0042] The medium for fusion cells was changed halfway every 2-4 days (discarding 100 μL and adding 100 μL of fresh HAT medium). Observation was performed after 7-8 days, and surviving wells were marked. When hybridoma cells reached more than 1 / 10 of the bottom of the well, the supernatant was collected and positive wells were screened using ELISA. Cells from positive wells were transferred to 24-well plates, counted after confluence, diluted to 10 cells / mL, and seeded into 96-well plates containing feeder cells. Screening was repeated. A portion of the cells was cryopreserved. Subcloning was performed three times consecutively until highly reactive monoclonal hybridoma cells were selected. High-titer, highly reactive monoclonal hybridoma cells were propagated and cryopreserved.

[0043] 1.4 Preparation and Reactivity Identification of Monoclonal Antibodies

[0044] BALB / c female mice aged 10–12 weeks were pre-stimulated by intraperitoneal injection of 0.5 mL Freund's incomplete adjuvant. Seven days later, the selected monoclonal hybridoma cell line was immunized into the mice at a dose of 3 × 10⁻⁶. 6 Cells / mouse. Ascites fluid was collected 7–10 days post-immunization, when the mice's abdomens were distended, and purified using a Protein G column. Purification results are shown below. Figure 1 As shown, lanes 1, 2, and 3 are for mouse ascites, purified flow-through, and purified washing, respectively, while lanes 4-6 are for the purified product, which is a monoclonal antibody prepared against type O foot-and-mouth disease inactivated virus, named monoclonal antibody 3G10.

[0045] The purified O / Cathay type foot-and-mouth disease virus inactivated antigen was electrophoresed, transferred onto a PVDF membrane, and subjected to Western blotting with the cell supernatant of monoclonal antibody 3G10 to verify reactivity. Results are as follows: Figure 2 As shown, the monoclonal antibody 3G10 specifically reacts with the 146S protein of O / Cathay type foot-and-mouth disease inactivated virus.

[0046] Purified O / Cathay type foot-and-mouth disease virus inactivated antigen was coated into ELISA plates at 300 ng per well and incubated overnight at 4°C. After washing with PBST, commercial blocking buffer was added and the plates were blocked overnight. The previously prepared monoclonal antibody 3G10 was serially diluted and added to the coated plates, incubated at 37°C for 1 h, and washed 4 times with PBST. HRP-labeled goat anti-mouse IgG (1:15000 dilution) was added, incubated at 37°C for 1 h, and washed 4 times with PBST. TMB was developed for 15 min, and then stop buffer was added. OD values ​​were read using a microplate reader. 450nm The titer of the monoclonal antibody 3G10 was determined. The results are as follows: Figure 3 As shown, the titer of monoclonal antibody 3G10 is greater than 1:128000.

[0047] The isotype of monoclonal antibody 3G10 was determined using a commercially available monoclonal antibody isotype identification kit. Results are as follows: Figure 4 As shown, the heavy chain of monoclonal antibody 3G10 is of type IgG2a, and the light chain is of type κ.

[0048] Foot-and-mouth disease virus (FMD) of types O / Cathay (O-GXCX-CHA-2018-S), O / MYA98, O / BY / 2010, OZK / 93, AF / 72, A / GDMM / 2013, and A / WH / 09 were coated onto ELISA plates, and the specificity of monoclonal antibody 3G10 was detected using an indirect ELISA method. Results are as follows: Figure 5 As shown, the monoclonal antibody 3G10 described in this application can specifically bind to foot-and-mouth disease virus O-GXCX-CHA-2018-S. This indicates that the monoclonal antibody 3G10 described in this application specifically reacts with O / Cathay type foot-and-mouth disease virus and has high specificity.

[0049] Example 2: Application of monoclonal antibodies in the quantitative detection of O / Cathay type foot-and-mouth disease virus typing

[0050] 2.1 Establishment of a quantitative detection method for foot-and-mouth disease virus (FMD) type O / Cathay

[0051] 2.1.1 Optimization of working conditions for the detection method

[0052] According to HRP Conjugation Kit-Lighting- The kit procedure involves HRP labeling the purified 3G10 capture antibody. The immobilized enzyme-labeled monoclonal antibody 3G10 is diluted 1:15000. The optimal operating conditions for the ELISA method were determined using checkerboard titration: 100 ng of capture antibody protein coating per well and a 3G10 dilution ratio of 1:30000.

[0053] 2.1.2 Establishment of the Standard Curve and Result Calculation Method

[0054] Standard curve establishment: O / Cathay type 146S antigen was serially diluted 2-fold to prepare standard solutions with concentrations ranging from 1 to 600 μg / mL. ELISA detection was performed using the screening conditions described above. The standard solutions were measured six times at different times and by different personnel. The measured OD values ​​were then analyzed. 450nm The values ​​were fitted to the theoretical concentration of the reference diluted product, and the coefficient of determination (R²) was calculated. R² > 0.99, indicating a good fit. A standard curve was then plotted, with the range determined to be 0.3–2.3, based on its OD... 450nm With the x-axis representing the values ​​and the y-axis representing the corresponding concentrations of the standards, plot the standard regression curve and equation: Left-click to select the data area, click "Insert" at the top of Excel, run the Chart Wizard to insert a "Scatter Plot xy (with data markers only)," and click OK. The chart will appear. Then right-click on the standard points in the chart and select "Add Trendline," checking "Polynomial, Show Formula, Show R-squared." This will give you the regression curve and equation in the chart. To ensure the detection of OD after gradient dilution of the positive standards... 450nm Within the specified range, the range corresponding to the antigen concentration (0.1875 μg / ml to 3 μg / ml) was selected based on this standard curve, and the initial concentration of the positive standard was determined to be 6 μg / ml.

[0055] Result calculation method: Select OD 450nm Substituting the OD values ​​(x values) of samples between 1.0 and 2.0 into the regression equation yields the y values ​​for samples at different dilutions. Multiplying the y values ​​by the corresponding dilution factor gives the sample concentration C. Finally, calculating the average C values ​​for the selected samples gives the antigen concentration for that sample. The calculation method is described in [link to calculation]. Figure 6 .

[0056] 2.2 Validation of the O / Cathay type foot-and-mouth disease virus O-type quantitative detection method

[0057] Accuracy verification: Foot-and-mouth disease virus O / Cathay type 146S antigen was diluted with PBS to three concentrations: high (600 μg / ml), medium (60 μg / ml), and low (6 μg / ml). The established method was used for detection. Reference samples at each concentration were tested three times, with two replicates per test. The recovery rate was calculated using the formula [Recovery / % = (Detected value / True value) × 100%]. Results are as follows: Figure 7 As shown, the recovery rate of this method for both antigens is above 95%, and the detection accuracy is high.

[0058] Repeatability validation: Four ELISA plates from different batches were taken, and the O / Cathay type 146S antigen was diluted with PBS to three concentrations: high (600 μg / ml), medium (60 μg / ml), and low (6 μg / ml). The established method was used for each concentration, with four replicates per sample. Intra-batch replicates were performed on the same ELISA plate, and inter-batch replicates were performed between different ELISA plates. The coefficient of variation was calculated based on the measured antigen concentration values. Results are as follows: Figure 8 As shown, the inter-assay and intra-assay coefficients of variation for the two antigens detected by this method are both less than 10%, indicating that the method has good reproducibility.

[0059] Specificity validation: The antigen content in foot-and-mouth disease viruses Re-O / MYA98, Re-O-GXCX-CHA-2018-S, AF / 72, A / GDMM / 2013, and A / WH / 09 was detected using the established detection procedure to validate the method's specificity. Results are as follows: Figure 9 As shown, the quantitative detection method for O / Cathay type foot-and-mouth disease virus antigens described in this invention does not exhibit cross-reactivity with other topological types of O and type A foot-and-mouth disease virus antigens, indicating that this method has good specificity.

[0060] Example 3: Sequencing of the variable region of a monoclonal antibody

[0061] 3.1 Extraction of total mRNA from positive hybridoma cells

[0062] Revive the previously frozen monoclonal hybridoma cells, culture for 2-3 generations, and prepare a cell suspension. Add 1 mL of TRizol, mix well, and incubate at 4°C for 5 min. Add 250 μL of chloroform, mix well, incubate at 4°C for 10 min, and centrifuge at 12000 rpm for 15 min. Take 450 μL of the supernatant, add an equal volume of isopropanol, incubate at -20°C for 30 min, and centrifuge as before. Discard the supernatant, wash the precipitate with 1 mL of 75% ethanol, and centrifuge for 5 min. Dry the precipitate, dissolve it in 25 μL of RNase-free water, and obtain total mRNA.

[0063] 3.2 cDNA Synthesis (Double-Strand) and Purification

[0064] Add the components shown in Table 1 to a 0.2 mL PCR amplification tube in sequence, mix well, place the PCR amplification tube in a PCR instrument, and amplify using the reaction program in Table 2. The product obtained is the whole genome cDNA, which should be stored at -20℃ for later use.

[0065] Table 1 Reverse transcription reagent system

[0066]

[0067] 3.3 Sequencing of the variable region of monoclonal antibodies

[0068] 3.2.1 Primer Synthesis

[0069] Reference (von Boehmer L, Liu C, Ackerman S, Gitlin AD, Wang Q, Gazumyan A, Nu ssenzweig MC. Sequencing and cloning of antigen-specific antibodies from mouse emery B cells. Nat Protoc. 2016 Oct; 11(10):1908-1923. doi:10.1038 / nprot.2016.102.Epub2016Sep15.PMID:27658009.) Primers as shown in SEQ ID No. 11-40 were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. Premixed primers were prepared according to the literature, and a PCR amplification program was designed for PCR amplification. The specific amplification primers are as follows:

[0070] First-round amplification primers:

[0071] (1) Primers for amplifying the VH sequence:

[0072] I: 5'-AGGAACTGCAGGTGTCC-3' (SEQ ID No. 11);

[0073] II: 5'-CAGCTACAGGTGTCCACTCC-3' (SEQ ID No. 12);

[0074] III: 5'-TGGCAGCARCAGCTACAGG-3' (SEQ ID No. 13);

[0075] IV: 5'-CTGCCTGGTGACATTCCCA-3' (SEQ ID No. 14);

[0076] V: 5'-CCAAGCTGTGTCCTGTC-3' (SEQ ID No. 15);

[0077] VI: 5'-TTTTAAAAGGTGTCCAGKGT-3' (SEQ ID No. 16);

[0078] VII: 5'-CCTGTCAGTAACTRCAGGTGTCC-3' (SEQ ID No. 17);

[0079] VIII: 5'-TTTTAAAGGGGTCCAGTGT-3' (SEQ ID No. 18);

[0080] IX: 5'-CGTTCCTGGTATCCTGTCT-3' (SEQ ID No.19);

[0081] X: 5'-ATGAAGTTGTGGYTRAACTGG-3' (SEQ ID No.20);

[0082] XI: 5'-TGTTGGGGCTKAAGTGGG-3' (SEQ ID No.21);

[0083] XII: 5'-AGAAGGTGTGCACACCGCTGGAC-3' (SEQ ID No.22).

[0084] (2) Primers for amplifying the VL sequence:

[0085] I: 5'-RGTGCAGATTTTCAGCTTCCTGCT-3' (SEQ ID No.23);

[0086] II: 5'-TGGACATGAGGGCYCCTGCTCAGT-3' (SEQ ID No.24);

[0087] III: 5'-CTSTGGTTGTCTGGTGTTGAYGGA-3' (SEQ ID No.25);

[0088] IV: 5'-GTTGCTGCTGCTGTGGCTTACA-3' (SEQ ID No.26);

[0089] V: 5'-GTATCTGGTACCTGTGG-3' (SEQ ID No.27);

[0090] VI: 5'-TGCCTGTTAGGCTGTTGGTGCT-3' (SEQ ID No.28);

[0091] VII: 5'-GCTCAGTTCCTTGGTCTCCTGTTGC-3' (SEQ ID No.29);

[0092] VIII: 5'-TGGGTGCTGCTGCTCTGGGT-3' (SEQ ID No.30);

[0093] IX: 5'-CAGTTCCTGTTTCTGTTARTGCTCTGG-3' (SEQ ID No.31);

[0094] X: 5'-TGCTCTGGTTATATGGTGCTGATGGG-3' (SEQ ID No. 32);

[0095] XI: 5'-ACTGAGGCACCTCCAGATGTT-3' (SEQ ID No. 33).

[0096] Second round amplification primers:

[0097] Ⅰ: 5'-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG-3' (SEQ ID No. 34); Ⅱ: 5'-GCTCAGGGAARTAGCCCTTGAC-3' (SEQ ID No. 35);

[0098] III: 5'-GAYATTGTGMTSACCMCARWCTMCA-3' (SEQ ID No. 36);

[0099] Ⅳ: 5'-TGGGAAGATGGATACAGTT-3' (SEQ ID No. 37);

[0100] V: 5'-CAGGCTGTTGTGACTCAG-3' (SEQ ID No. 38);

[0101] VI: 5'-CAACTTGTGCTCACTCAG-3' (SEQ ID No. 39);

[0102] VII: 5'-CTCYTCAGRGGAAGGTGGRAACA-3' (SEQ ID No. 40).

[0103] 3.2.2 First Round of Amplification

[0104] Add the components listed in Tables 3 and 4 to a 0.2 mL PCR amplification tube in sequence, and perform PCR amplification using the reaction program in Table 5. Store the amplification products at -20℃.

[0105] Table 3 Antibody VH gene PCR amplification system

[0106]

[0107] Table 4 Antibody VL PCR Amplification System

[0108]

[0109] Table 5 Antibody VH / VL Amplification Program

[0110]

[0111] 3.2.3 Second round of amplification

[0112] The VH / VL products from the first round of amplification were amplified again using the amplification system shown in Table 6, following the reaction procedures shown in Table 7. The amplified products were stored at -20℃.

[0113] Table 6 Antibody Second-Round PCR Amplification System

[0114]

[0115] Table 7. Antibody Second-Round PCR Amplification Procedure

[0116]

[0117] 3.3 Comparison of gene sequences

[0118] After amplification, the target fragment was ligated into the pMD-19T vector to construct a sequencing plasmid, which was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The variable region sequence obtained from sequencing was compared with the mouse antibody heavy chain variable region sequence, light chain Lamda chain, and light chain Kappa chain published on the NCBI website using the NCBI and IMGT gene banks.

[0119] Sequencing results showed that the amplified sequences were the complementarity-determining regions (CDRs) of the heavy and light chain variable regions of the monoclonal antibody, as shown in Table 8; the amino acid sequences of the light and heavy chain variable regions are shown in Table 9; and the gene sequences encoding the light and heavy chain variable regions are shown in Table 10.

[0120] Table 8. Complementation-determining region sequence of antibody variable region

[0121]

[0122] Table 9. Amino acid sequence of antibody variable region

[0123]

[0124] Table 10 Antibody Variable Region Base Sequence

[0125]

[0126] In summary, this invention provides a monoclonal antibody 3G10 for O / Cathay type foot-and-mouth disease virus. This monoclonal antibody specifically recognizes and binds to the 146S region of O / Cathay type foot-and-mouth disease virus, exhibiting advantages such as high specificity, sensitivity, and low preparation cost. A specific implementation example demonstrates the application of monoclonal antibody 3G10 in a serotyping and quantitative detection method for O / Cathay type foot-and-mouth disease virus, providing a reference for post-vaccine quality control and efficacy evaluation, and offering new technical support for controlling the spread of foot-and-mouth disease. The monoclonal antibody sequence provided by this invention can be used for recombination and modification using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity.

[0127] The embodiments described above are only some embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the patent claims of the present invention should be included within the scope of the patent claims of the present invention.

Claims

1. A monoclonal antibody against O / Cathay type foot-and-mouth disease virus, wherein the O / Cathay type foot-and-mouth disease virus monoclonal antibody binds to the 146S protein of O / Cathay type foot-and-mouth disease virus, characterized in that, The O / Cathay type foot-and-mouth disease virus monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 1, CDR2 shown in SEQ ID No. 2, and CDR3 shown in SEQ ID No. 3; The variable region CDR of the antibody light chain includes amino acid sequences such as CDR1 shown in SEQ ID No. 4, CDR2 shown in SEQ ID No. 5, and CDR3 shown in SEQ ID No.

6.

2. The O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1, characterized in that, The O / Cathay type foot-and-mouth disease virus monoclonal antibody includes an antibody heavy chain variable region with an amino acid sequence as shown in SEQ ID No. 7 and an antibody light chain variable region with an amino acid sequence as shown in SEQ ID No.

8.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2.

4. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 3.

5. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. The application of the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2 in the preparation of products for the quantitative detection of O / Cathay type foot-and-mouth disease virus typing.

7. The application of the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2 in the quantitative detection of O / Cathay type foot-and-mouth disease virus for non-disease diagnosis purposes.

8. A quantitative detection kit for O / Cathay type foot-and-mouth disease virus typing, characterized in that, The kit includes the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2.

9. The method for preparing the O / Cathay type foot-and-mouth disease virus monoclonal antibody as described in claim 1 or 2, characterized in that, The method is as follows: the heavy chain variable region and light chain variable region sequences of the O / Cathay type foot-and-mouth disease virus monoclonal antibody are linked to the antibody heavy chain constant region and light chain constant region sequences, respectively, and inserted into the expression vector to obtain the antibody heavy chain and antibody light chain recombinant expression plasmid; the obtained recombinant expression plasmid is co-transfected into expression cells, and O / Cathay type foot-and-mouth disease virus monoclonal antibody is obtained by culturing.

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