Anti-African swine fever virus P150 protein monoclonal antibody 20D5 and its application

Abstract

The present invention discloses anti-African swine fever virus P150 protein monoclonal antibody 20D5 and its application, belonging to the technical field of monoclonal antibodies; the present invention also discloses the sequence of African swine fever virus monoclonal antibody secreted by the hybridoma cell line 20D5. The hybridoma cell line 20D5 provided by the present invention and the monoclonal antibody secreted by it can react specifically with ASFV and ASFV-P150 protein, have good immunogenicity, good specificity and high sensitivity, can be widely used in immunoassay technology, can be used as antibodies for Western-Blot, indirect immunofluorescence and other tests, for identifying ASFV, ASFV-P150 protein and determining ASFV virus content. It can be seen that the present invention lays an important foundation for the basic research of ASFV and its P150 protein and the clinical detection of antigens.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to anti-African swine fever virus P150 protein monoclonal antibody 20D5 and its application. Background Art

[0002] African swine fever (ASF) is an acute, widespread hemorrhagic, and highly contagious disease caused by the African swine fever virus (ASFV) infecting pigs. Pigs of all ages are susceptible, and the mortality rate after onset can be as high as 100%. The viral genome ranges from 170 to 193 kbp, encoding approximately 68 structural proteins and more than 100 non-structural proteins. Mature virus particles ( Figure 1 ) is icosahedral symmetric, with a diameter of about 200nm. It is mainly composed of five parts, from the inside to the outside: the viral genome (Genome), inner core shell (Core shell), inner membrane (Inner envelope), capsid (capsid) and external envelope (External envelope).

[0003] To date, ASFV has been found to encode more than 150 ORFs in its viral genome. One of the ORFs encodes the ASFV polyprotein precursor PP220, which is essential for the structural integrity of the virus. PP220, with a relative molecular weight of 220.281 kDa, is a late protein in viral infection, located in the viral core shell, encoded by the gene CP2475L. PP220 (pCP2475L) and PP62 (pCP530R) are two major polyproteins, which are cleaved into mature structural proteins to participate in the maturation of viral particles. The PP220 polyprotein is initially processed into P150 protein and PP90 preprotein. The latter is cleaved into P34 protein and precursor PP55 protein, from which P5, P14 and P37 proteins are eventually generated.

[0004] The disease caused by ASFV is extremely harmful to the pig farming industry. Immunoassay technology can effectively detect the effective antigens of ASFV. For immunoassay technology, antibodies that can effectively bind to viral antigens are the key. Therefore, providing an antibody that can efficiently bind to ASFV or its antigens is an urgent problem to be solved in the field. In view of this, the present invention is specially proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-African swine fever virus P150 protein monoclonal antibody 20D5 and its application to solve the problems existing in the above-mentioned prior art. The hybridoma cell line 20D5 and the monoclonal antibody secreted by it can react specifically with ASFV and ASFV-P150 protein, have good specificity and high sensitivity, and can be used as antibodies for Western-Blot and indirect immunofluorescence tests to identify ASFV and P150 protein and determine the ASFV virus content. It can be seen that the present invention lays an important foundation for the clinical detection of ASFV and its P150 protein antigen.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a monoclonal antibody 20D5 against African swine fever virus P150 protein, wherein the heavy chain variable region of the monoclonal antibody 20D5 comprises a CDR1 with an amino acid sequence of SGYYWN, a CDR2 with an amino acid sequence of YISYDSSNNYNPSLKN, and a CDR3 with an amino acid sequence of ATGSAY; the light chain variable region of the monoclonal antibody 20D5 comprises a CDR1 with an amino acid sequence of KASQDVTTAVA, a CDR2 with an amino acid sequence of SASYRYT, and a CDR3 with an amino acid sequence of QPHFTHPPT.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 20D5 is shown in SEQ ID NO:1; and the amino acid sequence of the light chain variable region of the monoclonal antibody 20D5 is shown in SEQ ID NO:2.

[0009] Furthermore, the gene sequence of the heavy chain variable region of the monoclonal antibody 20D5 is shown in SEQ ID NO:3; the gene sequence of the light chain variable region of the monoclonal antibody 20D5 is shown in SEQ ID NO:4.

[0010] Furthermore, the African swine fever virus monoclonal antibody produces a specific immune response with the African swine fever virus or its P150 protein antigen.

[0011] The present invention also provides a gene encoding the monoclonal antibody 20D5, wherein the gene comprises a heavy chain variable region gene and a light chain variable region gene; the sequence of the heavy chain variable region gene is shown in SEQ ID NO:3; the sequence of the light chain variable region gene is shown in SEQ ID NO:4.

[0012] The present invention also provides a recombinant expression vector comprising the gene.

[0013] Furthermore, the above-mentioned recombinant expression vector is constructed using conventional genetic engineering technology.

[0014] The present invention also provides the use of the monoclonal antibody 20D5, the gene or the recombinant expression vector in preparing a detection product for African swine fever virus or its P150 protein.

[0015] Furthermore, the detection product comprises a reagent or a kit for detection using Western-Blot or indirect immunofluorescence method.

[0016] The present invention also provides a product for detecting African swine fever virus or its P150 protein antigen, wherein the product comprises the monoclonal antibody 20D5.

[0017] Furthermore, the product comprises reagents or kits for detection using Western-Blot or indirect immunofluorescence methods.

[0018] The present invention discloses the following technical effects:

[0019] The monoclonal antibody 20D5 secreted by the hybridoma cell line 20D5 provided by the present invention can react specifically with ASFV and ASFV-P150 proteins, has good immunogenicity, good specificity and high sensitivity, can be widely used in immunoassay technology, and can be used as an antibody for Western-Blot and indirect immunofluorescence test for identifying ASFV and P150 proteins and determining the ASFV virus content; at the same time, the present invention is obtained by conventional genetic engineering or protein engineering methods, which avoids the loss of antibody genes in long-term cryopreservation of hybridoma cells, and is also conducive to optimizing antibodies at the gene and protein levels, thereby improving the specificity and affinity of antibodies. It can be seen that the present invention lays an important foundation for the clinical detection of ASFV and its P150 protein antigen and the study of viral pathogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the ELISA result diagram of the reaction between the inactivated ASFV virus and the African swine fever virus-positive pig serum in clinical practice in Example 1; wherein the P30 protein antigen is a positive control;

[0022] Figure 2 The ELISA test results of serum antibody titer of immunized mice in Example 1;

[0023] Figure 3This is the result of IFA analysis of ASFV P150 monoclonal antibody in Example 2 (virus-infected cells);

[0024] Figure 4 The figure is the result of Western-Blot analysis of ASFV P150 monoclonal antibody in Example 2, M: Marker, 1: virus-infected cell sample, 2: blank cell control sample;

[0025] Figure 5 This is the electrophoresis diagram for amplification verification of the African swine fever P150 eukaryotic expression plasmid in Example 2;

[0026] Figure 6 This is a diagram of the ASFV P150 monoclonal antibody IFA analysis results in Example 2 (P150 protein expressing cells);

[0027] Figure 7 The figure is the result of Western-Blot analysis of ASFV P150 monoclonal antibody in Example 2, M: Marker, 1: pcaggs-p150 eukaryotic expression cell sample, 2: blank cell control sample;

[0028] Figure 8 This is a SDS-PAGE test result diagram of the purification effect of the ASFV P150 monoclonal antibody in Example 3;

[0029] Fig. 9 The results of PCR amplification of the variable regions in Example 4, M: Marker, VL: light chain of monoclonal antibody 20D5, VH: heavy chain of monoclonal antibody 20D5. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] 1 Screening and identification of monoclonal antibodies against ASFV P150 protein

[0037] 1.1 ASFV inactivated virus particle concentration

[0038] The ASFV virus solution inactivated by formaldehyde was added to the ultracentrifuge tube, and a 30% (mass-to-volume ratio) sucrose solution was added to the bottom of the ultracentrifuge tube (roughly to the arc position) using a long syringe. After balancing, it was concentrated using an ultrahigh-speed refrigerated centrifuge (Beckman Optima XE-90), dissolved in PBS, and the protein content was determined using Nano Drop.

[0039] 1.2 Verification of the reactivity of concentrated virus particles - indirect ELISA test

[0040] 10 μg / well of the concentrated inactivated virus in 1.1 and 1 μg / well of the P30 protein stored in the laboratory were coated on the ELISA plate at 4°C overnight, 5% skimmed milk powder was used to block the ELISA plate at room temperature for 4 h, and the ELISA plate was washed with PBST buffer. The immunized mouse serum was diluted from 1:40 and used as the primary antibody for incubation at room temperature for 2 h. The ELISA plate was washed with PBST buffer to wash away the unbound primary antibody, and HRP-labeled goat anti-mouse was diluted 1:6000 as the secondary antibody for incubation at room temperature for 45 min. The ELISA plate was washed with PBST buffer, 100 μL / well of the color developing solution was added and incubated at 37°C incubator for 10 min, and then 50 μL / well of the stop solution was added, and the results were observed by a UV spectrophotometer at 450 nm.

[0041] Figure 1 The results of ELISA experiments showed that the inactivated virus had good reactogenicity and could be used as an immunogen to immunize mice.

[0042] 1.3 Immunization of mice

[0043] The concentrated inactivated virus was emulsified with Freund's complete adjuvant at an equal volume of 1:1 at 100 μg / mouse and immunized by subcutaneous injection at multiple points on the back of mice. Two weeks later, Freund's incomplete adjuvant was emulsified with the inactivated virus at an equal volume of 1:1 and immunized once. The immunization was repeated once again after an interval of 2 weeks for a total of 3 immunizations. Fifteen days after the third immunization, blood was collected from the tail vein of the immunized mice.

[0044] 1.4 Indirect ELISA to detect serum titers of immunized mice

[0045] The ELISA plate was coated with 10 μg / well of inactivated viral protein at 4°C overnight, and the ELISA plate was blocked with 5% skim milk powder at room temperature for 4 hours. The ELISA plate was washed with PBST buffer, and the immunized mouse serum was diluted from 1:100 to 1:12800 as the primary antibody and incubated at room temperature for 2 hours. The ELISA plate was washed with PBST buffer to wash away the unbound primary antibody, and HRP-labeled goat anti-mouse was diluted 1:6000 as the secondary antibody and incubated at room temperature for 45 minutes. The ELISA plate was washed with PBST buffer, 100 μL / well of color development solution was added and incubated in a 37°C incubator for 10 minutes, and then 50 μL / well of stop solution was added, and the results were observed by a 450nm UV spectrophotometer. Figure 2 The results of the ELISA experiment showed that after mice were immunized with inactivated ASFV virus, antibodies against African swine fever virus protein were detected in the mouse serum 15 days after the third immunization, and the subsequent cell fusion test could be carried out.

[0046] 1.5 Cell fusion

[0047] The spleen cells of mice after booster immunization were taken and fused with sp2 / 0 cells at a ratio of (10-15):1 in a 37°C water bath. PEG4000 was added for 1 min, and the cells were allowed to stand for 1 min. Then, 1 mL of 1640 medium was added for 1 min, and 1 mL of 1640 medium was added for 20 s. Then, 30 mL of medium was added for 3 min. The cells were centrifuged at 800 rpm for 5 min, and 1×HAT 20% FBS 1640 medium was used for plating. When the confluence of the successfully fused cells reached more than 50%, positive cell lines were screened.

[0048] 1.6 Hybridoma cell screening and cloning

[0049] LLC-PK1 cells inoculated with ASFV virus solution were used for indirect immunofluorescence test, and the cell culture supernatant 10 days after fusion was collected for indirect immunofluorescence test to screen positive cell clones. Finally, hybridoma cell wells that reacted positively with ASFV infected cells and negatively with LLC-PK1 blank cells were selected, and subcloned by limiting dilution method. After two subclones and indirect immunofluorescence test screening, a hybridoma cell line 20D5 that stably secreted monoclonal antibodies against African swine fever virus protein was obtained.

[0050] Example 2

[0051] Identification of Antibodies Secreted by Hybridoma Cell Lines

[0052] 2.1 Indirect fluorescence immunoassay to identify the reactivity of fused hybridoma cells with ASFV

[0053] LLC-PK1 cells were digested with trypsin and plated in 24-well cell plates. When the cells grew to about 90% confluency, ASFV virus solution was inoculated and incubated at 37°C for 2 hours. Fresh 2% DMEM was then used to continue culturing. After 36 hours of culturing at 37°C, indirect immunofluorescence assay was performed. After discarding the cell culture medium, wash twice with PBS, add 4% paraformaldehyde to fix at room temperature for 15 minutes, discard the fixative, wash 3 times with PBS, use PBS containing 0.1% Triton 100 as permeabilization solution, permeabilize at room temperature for 15 minutes, discard the permeabilization solution, wash 3 times with PBS, change to PBS containing 5% BSA as blocking solution, block at room temperature for 2 hours, discard the blocking solution, add hybridoma supernatant as primary antibody, incubate at room temperature for 2 hours, wash 3 times with PBST, 5 minutes each time, add Alexa Flour 488 donkey anti-mouse IgG (H+L), 1:500 dilution, incubate at room temperature for 45 minutes, wash 3 times with PBST, 5 minutes each time, add ready-to-use DAPI to stain the cell nucleus for 5 minutes, discard DAPI, add PBS to wash 3 times, and observe the results under a fluorescence microscope. Observation under an inverted fluorescence microscope shows that the virus ASFV reacts specifically with the monoclonal antibody. After adding the fluorescent secondary antibody, obvious specific fluorescence appears under the field of view. The results are as follows Figure 3 shown.

[0054] 2.2 Western-blot identification of fusion hybridoma cells and ASFV reactivity

[0055] Collect LLC-PK1 cell samples 36 hours after inoculation with ASFV virus liquid, pre-treat and separate them by SDS-PAGE, take a piece of film containing the target band and place it on the membrane transfer instrument, and transfer the membrane at 200mA for 1.5 hours. Block the PVDF membrane with 10% skim milk at room temperature for 2 hours. Wash 3 times with PBST for 5 minutes each time, add hybridoma supernatant as primary antibody, and react slowly at 4°C overnight. Wash 3 times with PBST for 5 minutes each time, incubate with HRP-labeled rabbit anti-mouse IgG secondary antibody (1:5000) at room temperature for 45 minutes, and finally develop with ECL to observe the results. Western-blot identification results are shown as follows Figure 4 As shown. The results showed that the lanes had target bands at about 180 kDa and 220 kDa, indicating that the prepared monoclonal antibodies were able to produce a specific immune response with the ASFV virus.

[0056] 2.3 Construction of African swine fever P150 eukaryotic expression plasmid

[0057] Using the African swine fever genome as a template, the P150 gene was amplified using specific primers (Table 1) with a length of 4749 bp. After double digestion with EcoRI and NheI, it was connected to the pcaggs vector. The plasmid construction was verified by plasmid sequencing and specific primer amplification identification (e.g. Figure 5 ).

[0058] Table 1 P150 amplification primers

[0059] Primer name sequence P150-F CCGGAATTCGCCACCATGGCCGATGAGCTAG P150-R CTAGCTAGCTAAAATCCGAATATCACTATCATACTG

[0060] 2.4 Indirect fluorescence immunoassay to identify the reactivity of fused hybridoma cells with ASFV-P150 protein

[0061] Since WB identified the size of virus-infected cell proteins as 180 kDa and 220 kDa, HEK-293T cells were transfected with the P150 protein eukaryotic vector expression plasmid, and an indirect immunofluorescence test was performed 28 hours later. The experimental steps are as follows: discard the cell culture medium and wash once with PBS, add 4% paraformaldehyde to fix at room temperature for 15 minutes, discard the fixative, wash 3 times with PBS, use PBS containing 0.1% Triton 100 as permeabilization solution, permeabilize at room temperature for 15 minutes, discard the permeabilization solution, wash 3 times with PBS, switch to blocking solution containing 5% BSA (PBS), block at room temperature for 2 hours, discard the blocking solution, add hybridoma cell culture supernatant and HA antibody as primary antibody, incubate at room temperature for 2 hours, wash 3 times with PBST, each time for 5 minutes, add Alexa Flour 488 donkey anti-mouse IgG (H+L), Alexa Flour 594 mouse anti-human IgG (H+L), diluted 1:500, incubate at room temperature for 45 minutes, wash 3 times with PBST, each time for 5 minutes, add ready-to-use DAPI to stain cell nuclei for 5 minutes, discard DAPI, add PBS to wash 3 times, and observe the results under a fluorescence microscope. Observation under an inverted fluorescence microscope showed obvious specific fluorescence under the field of view, indicating that ASFV P150 protein and 20D5 hybridoma supernatant had specific antigen-antibody reaction, and the blank control well had no fluorescence. Figure 6 shown.

[0062] 2.5 Western-blot identification of the reactivity of monoclonal antibodies with ASFV-P150 protein

[0063] Since the protein size identified by WB is 180kDa and above, HEK-293T cells were transfected with the P150 protein eukaryotic vector expression plasmid. After 28 hours, the cell samples were collected, pre-treated and separated by SDS-PAGE. A film containing the target band was placed on the transfer apparatus and transferred at 350mA for 1.5 hours. The PVDF membrane was blocked with 10% skim milk at room temperature for 2 hours. After washing with PBST three times, each time for 5 minutes, a monoclonal antibody was added as the primary antibody, and the reaction was slowly shaken at 4°C overnight. After washing with PBST three times, incubate with HRP-labeled rabbit anti-mouse IgG secondary antibody (1:5000) at room temperature for 45 minutes, and finally use ECL development to observe the results. The Western-blot identification results are shown as follows. Figure 7 As shown, the results showed that each lane had a target band at approximately 180 kDa, indicating that the 20D5 hybridoma supernatant could produce a specific immune response with the ASFV virus and ASFV-P150 protein.

[0064] Example 3

[0065] Preparation and purification of ascites

[0066] 3.1 Preparation of mouse ascites

[0067] This method is often used to obtain antibodies with higher titers and more. The specific method is: take 3 healthy female Balb / c mice aged 6-8 weeks, intraperitoneally inject 0.5mL of sterile incomplete Freund's adjuvant per mouse; 7-10 days later, intraperitoneally inoculate 1-2×10 6 Hybridoma cells. Before inoculation, discard the culture medium of the hybridoma cells, wash the cells twice with serum-free 1640 medium, wash away the serum protein as much as possible, and blow away the cells. Under sterile conditions, inject 0.5mL of cell suspension into the mouse peritoneal cavity. Hybridoma cells multiply in large numbers in the form of ascites tumors in the mouse peritoneal cavity. After 7 to 10 days, the mouse abdomen is obviously swollen and feels fluctuating when touched. At this time, the ascites can be collected. The ascites is collected in a centrifuge tube and centrifuged at 10,000 r / min for 10 minutes. The colorless transparent layer of liquid in the middle is the monoclonal antibody ascites.

[0068] 3.3 Purification of ascites

[0069] The protein was purified by affinity chromatography using MCE's HY-K0214 Protein G Agarose and then its purity was determined by SDS-PAGE electrophoresis. Figure 8 As shown, the PAGE results of the ascites purified antibody showed two bands: a heavy chain of 55 kDa and a light chain of 25 kDa, indicating a good purification effect.

[0070] 3.4. Identification of monoclonal antibody types and subclasses

[0071] Mouse Mecrodinal Antibody Isotyping Elisa Kit Kit (purchased from Proteintech) was used to identify the subtype of the monoclonal antibody according to the instructions. The identification results are shown in Table 2:

[0072] Table 2 Identification of monoclonal antibody types

[0073] Monoclonal antibodies IgG1 IgG2a IgG2b IgG2c IgG3 IgM Kappa Lambda 20D5 + - - - - - + -

[0074] Note: + indicates a positive reaction, - indicates a negative reaction.

[0075] As shown in Table 2, the heavy chain subtype of monoclonal antibody 20D5 is IgG1, and the light chain subtype is Kappa.

[0076] Example 4

[0077] PCR Amplification and Sequencing of the Variable Region Gene of P150 Protein Monoclonal Antibody 20D5

[0078] The RNA of monoclonal antibody 20D5 hybridoma cells was extracted and reversed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). The variable region gene of the antibody was amplified by nested PCR. First, the above cDNA was used as a template to amplify the variable region gene of the antibody using the first round of mouse antibody IgG and κ light chain primers. Then, the first round product was used as a template to amplify the variable region gene of the antibody using the second round of mouse antibody IgG and κ light chain primers. The PCR reaction system was: 2×PhantaFlash Master Mix (Vazyme) 25μL, Primer1 and Primer2 1μL each, cDNA 1μL, ddH 2 O was added to 50 μL. The reaction procedure was: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min. The primers for antibody variable region gene amplification refer to the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nature protocols 11, 1908-1923.). After amplification, agarose gel electrophoresis was performed. The heavy chain and kappa light chain variable region gene size of monoclonal antibody 20D5 was about 300 bp ( Fig. 9), gel cutting and recovery of the target fragment. The recovered target fragment was inserted into the pMD-18T blunt-end vector for sequence determination. The sequencing results were compared and analyzed on the NCBI IGBLAST website, and the sequencing results confirmed that the amplified sequences were DNA of the heavy chain variable region and light chain variable region of the monoclonal antibody. Specifically, the DNA sequence encoding the heavy chain variable region of the mouse anti-ASFV P150 protein monoclonal antibody 20D5 is shown in SEQ ID NO:3; the DNA sequence encoding the light chain variable region of the mouse anti-ASFV P150 protein monoclonal antibody 20D5 is shown in SEQ ID NO:4. The amino acid sequence of the heavy chain variable region of the mouse anti-ASFV P150 protein monoclonal antibody 20D5 is shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region of the mouse anti-ASFV P150 protein monoclonal antibody 20D5 is shown in SEQ ID NO:2. The amino acid sequences of the heavy chain and light chain variable regions CDR1, CDR2 and CDR3 of the monoclonal antibody 20D5 are shown in the following table.

[0079] Table 3 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy and light chain variable regions of monoclonal antibody 20D5

[0080] CDR1 CDR2 CDR3 Heavy chain SGYYW YISYDSSNNYNPSLKN ATGSAY Light chain KASQDVTTAVA SASYRYT QPHFTHPPT

[0081] The amino acid sequence of the heavy chain variable region (VH) and the amino acid sequence of the light chain variable region (VL) of monoclonal antibody 20D5 are as follows:

[0082] DVQLQESGPGLVKPSQSLSLTCSVTGYSLTSGYYWNWIRQFPGNNLEWMGYISYDSSN NYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYFCARATGSAYWGQGTLVTVSA(SEQ ID NO:1);

[0083] DIVMTQSHKFMSTSVGDRVSITCKASQDVTTAVAWYQQKPGQSPKLLIYSASYRYTGV PDRFTGSGSGTDFTFTISSVQAEDLAVYYCQPHFTTHPPTFGGGTKLEIK(SEQ ID NO:2);

[0084] The gene sequences of the heavy chain variable region and light chain variable region of monoclonal antibody 20D5 are as follows:

[0085] GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAAACCTTCTCAGTCTCTGTC

[0086] TCTCACCTGCTCTGTCACTGGCTACTCCCTCACCAGTGGTTATTACTGGAACTGGATCCG

[0087] GCAGTTTCCAGGAAACAACCTGGAATGGATGGGCTACATAAGCTACGACAGTAGCAATA

[0088] ACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGT

[0089] TTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAGCTACATATTTCTGTGCAAGAG

[0090] CAACTGGGTCTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 3);

[0091] GACATTTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGACTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTGCTGATTTACTCGGCATCCTACCGG TACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCCAATTTTACTCATCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO:4).

[0092] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Monoclonal antibody 20D5 against African swine fever virus P150 protein, characterized in that: The heavy chain variable region of the monoclonal antibody 20D5 includes a CDR1 with an amino acid sequence of SGYYWN, a CDR2 with an amino acid sequence of YISYDSSNNYNPSLKN, and a CDR3 with an amino acid sequence of ATGSAY; the light chain variable region of the monoclonal antibody 20D5 includes a CDR1 with an amino acid sequence of KASQDVTTAVA, a CDR2 with an amino acid sequence of SASYRYT, and a CDR3 with an amino acid sequence of QPHFTHPPT.

2. The monoclonal antibody 20D5 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 20D5 is shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region of the monoclonal antibody 20D5 is shown in SEQ ID NO:

2.

3. The monoclonal antibody 20D5 according to claim 1, characterized in that The gene sequence of the heavy chain variable region of the monoclonal antibody 20D5 is shown in SEQ ID NO:3; the gene sequence of the light chain variable region of the monoclonal antibody 20D5 is shown in SEQ ID NO:

4.

4. The monoclonal antibody 20D5 according to claim 1, characterized in that The African swine fever virus monoclonal antibody generates a specific immune response with the African swine fever virus or its P150 protein antigen.

5. The gene encoding the monoclonal antibody 20D5 according to claim 1, characterized in that: The genes include a heavy chain variable region gene and a light chain variable region gene; the sequence of the heavy chain variable region gene is shown in SEQ ID NO:3; the sequence of the light chain variable region gene is shown in SEQ ID NO:

4.

6. A recombinant expression vector comprising the gene according to claim 5.

7. Use of the monoclonal antibody 20D5 according to any one of claims 1 to 4, the gene according to claim 5 or the recombinant expression vector according to claim 6 in the preparation of a detection product for African swine fever virus or its P150 protein.

8. The use according to claim 7, characterized in that: The detection product is a reagent or kit for detection using Western-Blot or indirect immunofluorescence method.

9. A product for detecting African swine fever virus or its P150 protein antigen, characterized in that: The product comprises the monoclonal antibody 20D5 according to any one of claims 1 to 4.

10. The product according to claim 9, characterized in that The product is a reagent or kit for detection using Western-Blot or indirect immunofluorescence method.

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