Sat1 type foot-and-mouth disease virus monoclonal antibody 5g10, kit and detection method

Abstract

The application belongs to the technical field of biology and specifically relates to a SAT1 type foot-and-mouth disease virus monoclonal antibody 5G10, a kit and a detection method. The single-chain antibody 5G10 comprises 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. 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 7. The titer is greater than 1:128000, the heavy chain is IgG1 type, and the light chain is lambda type. The antibody can specifically combine with VP1 recombinant protein of the SAT1 type foot-and-mouth disease virus and has high specificity. The application of the monoclonal antibody 5G10 in a foot-and-mouth disease virus SAT1 type blocking ELISA antibody detection method is provided, and convenient and effective technical reserves are provided for border port detection and animal serum antibody screening of the SAT type foot-and-mouth disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a SAT1 type foot-and-mouth disease virus monoclonal antibody 5G10, a reagent kit, and a detection method. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, highly contagious viral disease of livestock caused by the foot-and-mouth disease virus (FMDV). It primarily affects cattle, pigs, sheep, and other cloven-hoofed ruminants, causing significant economic losses to the livestock industry. FMDV exhibits high genetic and antigenic variability, and seven serotypes have been identified: O, A, C, Asia1, SAT1, SAT2, and SAT3. There is no cross-protection between different serotypes, greatly increasing the difficulty of FMD prevention, control, and diagnosis.

[0003] African buffalo are the primary natural reservoir and sustaining host for SAT1, SAT2, and SAT3 serotypes. The virus continuously circulates and evolves within African buffalo populations, typically without causing severe illness in the buffalo, but it can spread to livestock, triggering serious outbreaks. Due to its long-term evolution in African buffalo and other wild animal populations, the SAT1 serotype possesses extremely rich genetic and antigenic diversity. Different lineages of SAT1 viruses exhibit significant antigenic differences, making the diagnosis of SAT1 foot-and-mouth disease virus extremely complex, requiring diagnostic methods (such as ELISA and PCR) capable of specifically distinguishing SAT1 from other serotypes. In outbreak investigations, VP1 gene sequencing is also necessary to determine its specific topology / lineage to trace the source of the outbreak. Therefore, providing a method or kit capable of specifically diagnosing SAT1 foot-and-mouth disease virus has become a pressing technical problem for those skilled in the art.

[0004] Monoclonal antibodies, due to their targeting of specific epitopes of antigens, possess high homogeneity, strong specificity, and high purity. The use of monoclonal antibodies to prepare viral detection kits, especially rapid test strips, is a successful example of transforming cutting-edge biotechnology into universally accessible public health products. Currently, there is no effective vaccine for SAT1 foot-and-mouth disease virus (FMDV) available in China. Therefore, in the absence of vaccine protection, establishing serological detection methods for SAT1 FMDV as a technical reserve for import and export port quarantine and border area prevention and control screening is crucial to prevent the introduction of SAT1 FMDV into my country. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a monoclonal antibody 5G10 against SAT1 foot-and-mouth disease virus and to establish a blocking ELISA antibody detection method for SAT1 type. This method enables serological diagnosis of SAT1 type foot-and-mouth disease virus, possessing high specificity, high sensitivity, and good reproducibility. It can accurately distinguish SAT1 type from other serotypes of FMDV infection antibodies, avoiding cross-reaction interference. Furthermore, the method is simple to operate, has a short detection time, and is easy to deploy and apply rapidly in border areas, making it of significant importance in SAT1 type foot-and-mouth disease serological antibody screening. Specifically, it includes the following:

[0006] In a first aspect, the present invention provides a SAT1 type foot-and-mouth disease virus monoclonal antibody 5G10, wherein the monoclonal antibody 5G10 includes a heavy chain variable region and a light chain variable region; the CDR of the heavy chain variable region includes amino acid sequences as shown in SEQ ID No. 1 CDR1, SEQ ID No. 2 CDR2 and SEQ ID No. 3 CDR3; the CDR of the light chain variable region includes amino acid sequences as shown in SEQ ID No. 4 CDR1 and SEQ ID No. 5 CDR3, wherein the amino acid sequence of CDR2 is WAS.

[0007] Preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 7.

[0008] Secondly, the present invention provides a gene fragment encoding the monoclonal antibody 5G10, wherein the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 8, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No. 9.

[0009] Thirdly, the present invention provides an expression vector containing the aforementioned gene fragment.

[0010] Fourthly, the present invention provides a host cell containing the expression vector described above, or having the gene fragment described above integrated into its genome.

[0011] Fifthly, the present invention provides the application of the monoclonal antibody 5G10 in the preparation of reagents, test strips, or kits for detecting murine SAT1 type foot-and-mouth disease virus.

[0012] In a sixth aspect, the present invention provides an antibody blocking ELISA detection kit for detecting SAT1 type foot-and-mouth disease virus, the detection kit comprising the monoclonal antibody described above.

[0013] Preferably, the detection kit further includes an ELISA plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

[0014] In a seventh aspect, the present invention provides the application of the aforementioned test kit in the detection of SAT1 type foot-and-mouth disease virus infection / immunity for non-disease diagnosis purposes.

[0015] Eighthly, the present invention provides a method for detecting serum antibodies against SAT1 type foot-and-mouth disease virus, comprising the following steps:

[0016] After performing blocking ELISA detection using the described blocking ELISA detection kit, the OD values ​​of the positive control, negative control, and samples were measured respectively. 450nm And calculate the blocking rate;

[0017] Using N as a negative control OD 450nm The average value, P is the positive control OD 450nm The average value, S is the OD of the tested sample. 450nm The average value;

[0018] Using B as the blocking rate, the blocking rate of the positive control ;

[0019] Blocking rate of the tested sample ;

[0020] The positive control was serum from animals immunized with SAT1 type foot-and-mouth disease recombinant VP1 protein, with a blocking rate >60% after blocking ELISA detection;

[0021] The negative control was serum from healthy, non-immunized pigs with foot-and-mouth disease, which underwent OD detection using a blocking ELISA. 450nm >1.0;

[0022] When the blocking rate of the tested sample is ≥40%, it proves that there is an antibody against SAT1 type foot-and-mouth disease virus.

[0023] Preferably, before performing blocking ELISA detection using the blocking ELISA detection kit, the method further includes enzyme labeling of the monoclonal antibody 5G10 to obtain an enzyme conjugate; the enzyme labeling includes horseradish peroxidase labeling.

[0024] The present invention has the following beneficial effects: (1) The present invention provides a SAT1 type foot-and-mouth disease virus monoclonal antibody 5G10, wherein the single chain antibody 5G10 includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID No. 6, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No. 7.

[0025] (2) The monoclonal antibody 5G10 has a titer greater than 1:128000, with the heavy chain being IgG1 type and the light chain being κ type; the monoclonal antibody 5G10 can specifically bind to the VP1 recombinant protein of SAT1 type foot-and-mouth disease virus. This indicates that the monoclonal antibody 5G10 described in this application specifically reacts with SAT1 type foot-and-mouth disease virus and has high specificity.

[0026] (3) The single-chain antibody of SAT1 foot-and-mouth disease virus prepared by the present invention fills the gap of SAT1 foot-and-mouth disease virus specific ScFv antibody in the current market, and provides a small molecule antibody with low immunogenicity and high specificity, providing new materials for the serotype identification, diagnosis and control of foot-and-mouth disease virus, and providing new technical support for the monitoring and screening of SAT1 foot-and-mouth disease.

[0027] (4) The present invention also provides an application of monoclonal antibody 5G10 in the detection method of SAT1 type foot-and-mouth disease virus blocking ELISA antibody, which provides a convenient and effective technical reserve for the border port detection of SAT type foot-and-mouth disease and the screening of animal serum antibodies. Attached Figure Description

[0028] 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.

[0029] Figure 1 Purification results of monoclonal antibody 5G10;

[0030] Figure 2 Western blot (WB) results of the reaction between monoclonal antibody 5G10 and recombinant VP1 protein of SAT1 type foot-and-mouth disease virus;

[0031] Figure 3 Results of titer assay for the prepared monoclonal antibody 5G10;

[0032] Figure 4 Results of 5G10 subtype identification of monoclonal antibody;

[0033] Figure 5 Specificity identification results of monoclonal antibody 5G10;

[0034] Figure 6 Establishment of the critical value for the detection method of foot-and-mouth disease virus SAT1 type blocking ELISA antibody. Detailed Implementation

[0035] The present invention will be described in detail below through specific embodiments, but 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.

[0036] 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.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1: Preparation of Antibody against Mouse-Derived SAT1 Type Foot-and-Mouth Disease Virus ScFv

[0039] 1. Immunizing mice

[0040] SAT1 recombinant foot-and-mouth disease 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. Blood samples were collected from the tail vein one week after the third immunization to measure antibodies. OD... 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.

[0041] 2. Cell fusion

[0042] 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 PEG 2000. 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.

[0043] 3. Screening and subcloning of positive hybridoma cells

[0044] The medium for fusion cells was changed every 2-4 days (discarding 100 μL and adding 100 μL of fresh HAT medium). After 7-8 days, the cells were observed and the surviving wells were marked. When the hybridoma cells reached more than 1 / 10 of the bottom of the well, the supernatant was collected and positive wells were selected 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. The selection process was repeated. A portion of the cells was cryopreserved. After three subclonings, if the supernatant from all wells remained positive, a cell line secreting monoclonal antibodies against SAT1 type foot-and-mouth disease virus was obtained.

[0045] 4. Extraction of total mRNA from positive hybridoma cells

[0046] Take 400 μL of positive hybridoma cell solution, add 1 mL of Rizol, 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 supernatant, add an equal volume of isopropanol, incubate at -20°C for 30 min, and centrifuge as above. 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 to obtain total mRNA. The concentration was measured to be 356 ng / μL, and the purity was OD0.05. 260 / OD 280 It is 1.87.

[0047] 5. Preparation and reactivity identification of monoclonal antibodies

[0048] 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.

[0049] Purification results are as follows Figure 1 As shown, lane 1 contains mouse ascites fluid, and lane 2 contains the purified product, which was prepared as a monoclonal antibody against SAT1 type foot-and-mouth disease inactivated virus, named monoclonal antibody 5G10.

[0050] The recombinant VP1 protein of SAT1 foot-and-mouth disease virus was electrophoresed, transferred onto a PVDF membrane, and subjected to Western blotting with cell supernatant containing monoclonal antibody 5G10 to verify reactivity. Results are as follows: Figure 2 As shown, the monoclonal antibody 5G10 specifically reacts with the VP1 recombinant protein of SAT1 type foot-and-mouth disease virus.

[0051] The recombinant VP1 protein of SAT1 foot-and-mouth disease virus was coated into ELISA plates at 100 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 prepared monoclonal antibody 3G10 was serially diluted and added to the coated plates. The plates were incubated at 37°C for 1 h and washed four times with PBST. HRP-labeled goat anti-mouse IgG (1:15000 dilution) was added and incubated at 37°C for 1 h. After washing four times with PBST, TMB was added for 15 min, and then stop buffer was added. The OD values ​​were read using a microplate reader. 450nm The titer of monoclonal antibody 3G10 was determined.

[0052] The results are as follows Figure 3 As shown, the titer of monoclonal antibody 5G10 is greater than 1:128000.

[0053] 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 5G10 is of type IgG1, and the light chain is of type λ.

[0054] The inactivated foot-and-mouth disease viruses of types O / MYA98, O / BY / 2010, OZK / 93, AF / 72, A / GDMM / 2013, and A / WH / 09 (all types of inactivated foot-and-mouth disease viruses of types O / MYA98, O / BY / 2010, OZK / 93, AF / 72, A / GDMM / 2013, and A / WH / 09 were provided by the National Foot-and-Mouth Disease Reference Laboratory of Lanzhou Veterinary Research Institute), and the VP1 recombinant protein of type SAT1 foot-and-mouth disease virus (prepared and preserved in our laboratory) were coated onto ELISA plates, and the specificity of the monoclonal antibody 5G10 was detected by indirect ELISA.

[0055] The results are as follows Figure 5 As shown, the monoclonal antibody 5G10 can specifically bind to the VP1 recombinant protein of SAT1 foot-and-mouth disease virus, indicating that the monoclonal antibody 5G10 described in this application has a specific reaction with SAT1 foot-and-mouth disease virus and has high specificity.

[0056] Example 2: Application of monoclonal antibody 5G10 in the detection method of foot-and-mouth disease virus SAT1 blocking ELISA antibody.

[0057] 1. Preparation of SAT1 type enzyme-labeled antibody working solution

[0058] 1.1 Preparation of enzyme-labeled antibodies

[0059] Add 0.3 ml of 5G10 antibody solution (4 mg / ml) and 0.1 ml of activated peroxidase to a 1.5 ml EP tube, gently pipette to mix, and incubate overnight at 4°C. Add 40 μl of triethanolamine to the mixture, pipette to mix, then add 50 μl of sodium borohydride, pipette to mix again, and incubate at 4°C for 30 minutes. Transfer 10 μl of glycine solution to the mixture and pipette to mix thoroughly. Transfer the mixture to a dialysis bag, place it in a beaker containing 200 ml of dialysis buffer, and incubate overnight at 4°C, changing the dialysis buffer 3 times during this period. Use a syringe to extract the mixture from the dialysis bag, add an equal volume of antibody protectant, pipette to mix, aliquot, and store below -70°C.

[0060] 1.2 Preparation of enzyme-labeled antibody working solution

[0061] The prepared 5G10-HRP enzyme-labeled antibody was diluted to 35 ng / ml with enzyme-labeled antibody dilution buffer, mixed well, aliquoted, and stored at 2~8℃.

[0062] 2. Positive and negative control sera

[0063] The positive control serum was serum from animals immunized with the SAT1 type foot-and-mouth disease virus VP1 recombinant protein. The negative control serum was serum from healthy, non-immunized pigs with foot-and-mouth disease virus (antibody titer not exceeding 1:8 as detected by the foot-and-mouth disease virus type O and A liquid-phase blocking ELISA antibody detection kit, and negative as detected by the non-structural protein 3ABC antibody detection kit). The positive control serum was negative for both type O and type A foot-and-mouth disease antibody liquid-phase blocking ELISA detection kits, indicating that the commercially available O and A type foot-and-mouth disease antibody detection kits cannot detect the SAT1 type foot-and-mouth disease virus recombinant protein immunized serum.

[0064] 3. Foot-and-mouth disease virus SAT1 blocking ELISA antibody detection method

[0065] 3.1 Determination of the optimal conditions for blocking ELISA detection method

[0066] Based on the principle of solid-phase blocking ELISA, the optimal antigen coating amount, serum dilution ratio, enzyme-labeled antibody working solution concentration, serum reaction time, and enzyme-labeled antibody reaction time were determined as follows: the optimal coating concentration of VP1 protein was 1.5 μg / ml, 100 μl / well; the optimal dilution ratio of the test serum was 1:8; the optimal working concentration of the enzyme-labeled antibody working solution was 50 ng / ml; the optimal reaction time of the test serum was 30 minutes; the optimal reaction time of the enzyme-labeled antibody was 30 minutes; the optimal reaction time of the substrate solution was 12 ± 1 minutes; and the optimal blocking solution was 0.5% BSA + 1% sucrose + 2% trehalose-PBS solution.

[0067] 3.2 Determination of criteria for blocking ELISA

[0068] One hundred and eighty foot-and-mouth disease virus (FMD) virus-negative sera (120 negative swine sera, 30 negative bovine sera, and 30 negative sheep sera), with a clear background and confirmed by neutralization tests at a foot-and-mouth disease reference laboratory, were diluted with sample diluent at the optimal dilution ratio. The OD of each serum sample was measured using the established ELISA method. 450nm Calculate the PI value: PI = (1 - sample OD) 450nm / Blank control OD 450nm ) × 100%, and calculate the average of all PI values ​​( ) and standard deviation (SD), expressed by the formula Cut-off = +3SD is used to calculate the critical value. Serum mean PI is calculated. =20.11% and standard deviation SD=7.31%, using the formula Cut-off= +3SD calculates the critical value Cut-off as 39.48%.

[0069] For ease of calculation, the judgment criteria for this method are defined as follows: when the tested serum PI ≥ 40%, it is considered positive for foot-and-mouth disease virus SAT1 antibody; when the tested serum PI < 40%, it is considered negative for foot-and-mouth disease virus SAT1 antibody. Results are shown below. Figure 6 .

[0070] 3.3 Blocking ELISA Procedure

[0071] Remove the ELISA plate and add 50 µl of the serum to be tested and positive / negative controls to each well, two wells per sample. Incubate at 37°C for 30 minutes. Discard the liquid from each well into the waste container. Add 300 µl of 1× washing buffer to each well and wash the wells, discarding the washing buffer. Repeat this washing process for 5 wells. After discarding the last washing buffer, pat the remaining washing buffer in the wells dry on absorbent paper. Add 50 µl of enzyme-labeled antibody working solution to each well, seal the plate with sealing film, and incubate at 37°C for 30 minutes. Repeat the washing steps above. Add 50 µl of substrate solution to each well and incubate at 37°C in the dark for 12 ± 1 minutes. Add 50 µl of stop solution to each well and incubate at OD500 of the ELISA reader. 450nm Read the OD value at the specified wavelength.

[0072] Results calculation: OD of the negative control wells was calculated separately. 450nm The average value N, and the OD of the positive control well. 450nm Mean value P, OD of the tested sample well 450nm Average value S;

[0073] Using B as the blocking rate, the blocking rate of the positive control ;

[0074] Blocking rate of the tested sample ;

[0075] Test validity: OD of negative control well 450nm The mean N ≥ 1.0, and the positive control blocking rate ≥60%.

[0076] Result determination: If the sample blocking rate ≥40% indicates the sample is positive for Foot-and-Mouth Disease Virus SAT1 antibody; if the sample blocking rate is ≥40%, the sample is considered positive for Foot-and-Mouth Disease Virus SAT1 antibody. <40% indicates that the sample was negative for Foot-and-Mouth Disease Virus SAT1 antibody.

[0077] Example 3: Sequencing of the variable region of a monoclonal antibody

[0078] 1. Extraction of total mRNA from positive hybridoma cells

[0079] 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.

[0080] 2. cDNA double-strand synthesis and purification

[0081] (1) Whole-genome cDNA synthesis

[0082] 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.

[0083] Table 1 Reverse transcription reagent system

[0084] Table 2 Reverse Transcription Procedure

[0085] 3. Determination of the variable region sequence of monoclonal antibodies

[0086] 3.1 Primer Synthesis

[0087] Primers as shown in SEQ ID No. 11-40 were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd. After preparing premixed solutions for each primer group, a PCR amplification program was designed, and PCR amplification reactions were performed. The specific amplification primers are as follows:

[0088] First-round amplification primers:

[0089] (1) Primers for amplifying the VH sequence:

[0090] I: 5'-AGGAACTGCAGGTGTCC-3' (SEQ ID No. 10)

[0091] II: 5'-CAGCTACAGGTGTCCACTCC-3' (SEQ ID No. 11)

[0092] III: 5'-TGGCAGCARCAGCTACAGG-3' (SEQ ID No. 12)

[0093] IV: 5'-CTGCCTGGTGACATTCCCA-3' (SEQ ID No. 13)

[0094] V: 5'-CCAAGCTGTGTCCTGTC-3' (SEQ ID No. 14)

[0095] VI: 5'-TTTTAAAAGGTGTCCAGKGT-3' (SEQ ID No. 15)

[0096] VII: 5'-CCTGTCAGTAACTRCAGGTGTCC-3' (SEQ ID No. 16)

[0097] VIII: 5'-TTTTAAAGGGGTCCAGTGT-3' (SEQ ID No. 17)

[0098] IX: 5'-CGTTCCTGGTATCCTGTCT-3' (SEQ ID No. 18)

[0099] X: 5'-ATGAAGTTGTGGYTRAACTGG-3' (SEQ ID No. 19)

[0100] XI: 5'-TGTTGGGGCTKAAGTGGG-3' (SEQ ID No. 20)

[0101] Ⅻ: 5'-AGAAGTGTGCACACCGCTGGAC-3' (SEQ ID No. 21)

[0102] (2) Primers for amplifying the VL sequence:

[0103] I: 5'-RGTGCAGATTTTCAGCTTCCTGCT-3' (SEQ ID No. 22)

[0104] II: 5'-TGGACATGAGGGCYCCTGCTCAGT-3' (SEQ ID No. 23)

[0105] III: 5'-CTSTGGTTGTCTGGTGTTGAYGGA-3' (SEQ ID No. 24)

[0106] IV: 5'-GTTGCTGCTGCTGTGGCTTACA-3' (SEQ ID No. 25)

[0107] V: 5'-GTATCTGGTACCTGTGG-3' (SEQ ID No. 26)

[0108] VI: 5'-TGCCTGTTAGGCTGTTGGTGCT-3' (SEQ ID No. 27)

[0109] VII: 5'-GCTCAGTTCCTTGGTCTCCTGTTGC-3' (SEQ ID No. 28)

[0110] VIII: 5'-TGGGTGCTGCTGCTCTGGGT-3' (SEQ ID No. 29)

[0111] IX: 5'-CAGTTCCTGTTTCTGTTARTGCTCTGG-3' (SEQ ID No. 30)

[0112] X: 5'-TGCTCTGGTTATATGGTGCTGATGGG-3' (SEQ ID No. 31)

[0113] Ⅺ: '-ACTGAGGCACCTCCAGATGTT-3' (SEQ ID No. 32)

[0114] Second round amplification primers:

[0115] Ⅰ: 5'-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG-3' (SEQ ID No. 33)

[0116] Ⅱ: 5'-GCTCAGGGAARTAGCCCTTGAC-3' (SEQ ID No. 34)

[0117] Ⅲ: 5'-GAYATTGTGMTSACCMCARWCTMCA-3' (SEQ ID No. 35)

[0118] Ⅳ: 5'-TGGGAAGATGGATACAGTT-3' (SEQ ID No. 36)

[0119] Ⅴ: 5'-CAGGCTGTTGTGACTCAG-3' (SEQ ID No. 37)

[0120] VI: 5'-CAACTTGTGCTCACTCAG-3' (SEQ ID No. 38)

[0121] Ⅶ: 5'-CTCYTCAGRGGAAGGTGGRAACA-3' (SEQ ID No. 39)

[0122] 3.2 First Round of Amplification

[0123] 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℃.

[0124] Table 3 Antibody VH gene PCR amplification system

[0125] Table 4. Antibody VL gene PCR amplification system

[0126] Table 5 Antibody VH / VL Amplification Program

[0127] 3.3 Second Round of Amplification

[0128] 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℃.

[0129] Table 6 Antibody Second-Round PCR Amplification System

[0130] Table 7. Antibody Second-Round PCR Amplification Procedure

[0131] 4. Comparison of gene sequences

[0132] 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.

[0133] 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.

[0134] Table 8. Complementation-determining region sequence of antibody variable region

[0135] Table 9. Amino acid sequence of antibody variable region

[0136] Table 10 Antibody Variable Region Base Sequence

[0137] In summary, this invention provides a murine SAT1 type foot-and-mouth disease virus monoclonal antibody 5G10. The single-chain antibody 5G10 includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID No. 6, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No. 7. The monoclonal antibody 5G10 has a titer greater than 1:128000, the heavy chain is IgG1 type, and the light chain is λ type. The monoclonal antibody 5G10 can specifically bind to the VP1 recombinant protein of SAT1 type foot-and-mouth disease virus. This indicates that the monoclonal antibody 5G10 of this application specifically reacts with SAT1 type foot-and-mouth disease virus and has high specificity. The SAT1 type foot-and-mouth disease virus single-chain antibody prepared by this invention fills the gap in the current market for SAT1 type foot-and-mouth disease virus-specific ScFv antibodies, providing a small molecule antibody with low immunogenicity and high specificity. It provides new materials for the serotype identification, diagnosis, and control of foot-and-mouth disease virus and provides new technical support for the monitoring and screening of SAT1 foot-and-mouth disease. This invention also provides the application of monoclonal antibody 5G10 in the detection method of SAT1 type foot-and-mouth disease virus blocking ELISA antibody, providing a convenient and effective technical reserve for the border port detection of SAT type foot-and-mouth disease and the screening of animal serum antibodies.

[0138] 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.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody 5G10 against SAT1 type foot-and-mouth disease virus, characterized in that, The monoclonal antibody 5G10 includes a heavy chain variable region and a light chain variable region; the CDR of the heavy chain variable region includes amino acid sequences as shown in SEQ ID No. 1 (CDR1), SEQ ID No. 2 (CDR2), and SEQ ID No. 3 (CDR3); the CDR of the light chain variable region includes amino acid sequences as shown in SEQ ID No. 4 (CDR1) and SEQ ID No. 5 (CDR3), and the amino acid sequence of CDR2 is WAS.

2. The monoclonal antibody 5G10 as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

7.

3. A gene fragment encoding the monoclonal antibody 5G10 as described in claim 2, characterized in that, The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 8, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID No.

9.

4. An expression carrier, characterized in that, The expression vector contains the gene fragment as described in claim 3.

5. A host cell, characterized in that, The host cell contains the expression vector of claim 4, or the gene fragment of claim 3 is integrated into its genome.

6. The use of the monoclonal antibody 5G10 as described in claim 1 or 2 in the preparation of reagents, test strips, or kits for detecting murine SAT1 type foot-and-mouth disease virus.

7. An antibody blocking ELISA kit for detecting SAT1 type foot-and-mouth disease virus, characterized in that, The kit comprises the monoclonal antibody as described in claim 1 or 2.

8. The application of the test kit as described in claim 7 in the detection of SAT1 foot-and-mouth disease virus infection / immunity for non-disease diagnosis purposes.

9. A method for detecting serum antibodies against SAT1 type foot-and-mouth disease virus, characterized in that, Includes the following steps: After performing a blocking ELISA test using the blocking ELISA detection kit described in claim 7, the OD values ​​of the positive control, negative control, and sample were detected respectively. 450nm And calculate the blocking rate; Using N as a negative control OD 450nm The average value, P is the positive control OD 450nm The average value, S is the OD of the tested sample. 450nm The average value; Using B as the blocking rate, the blocking rate of the positive control ; Blocking rate of the tested sample ; The positive control was serum from animals immunized with SAT1 type foot-and-mouth disease recombinant VP1 protein, with a blocking rate >60% after blocking ELISA detection; The negative control was serum from healthy, non-immunized pigs with foot-and-mouth disease, which underwent OD detection using a blocking ELISA. 450nm >1.0; When the blocking rate of the tested sample is ≥40%, it proves that there is an antibody against SAT1 type foot-and-mouth disease virus.

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