Anti-African swine fever virus P22 protein antibody and its application

Abstract

The present invention discloses a hybridoma cell, and its microbial deposit number is CCTCC NO: C2024215. The present invention also discloses a bELISA method, and its core steps are: coating the bottom surface of a microplate with African swine fever virus P22 protein, adding serum to be tested to the microplate, incubating the microplate with a monoclonal antibody against African swine fever virus P22 protein secreted by the hybridoma cell labeled with horseradish peroxidase, adding a TMB substrate colorimetric solution to the microplate, and measuring OD 450nm The value is used to determine whether the serum to be tested contains antibodies against the P22 protein of the African swine fever virus. This method has strong specificity and high sensitivity, can be used for the detection of antibodies to the African swine fever virus, and has clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products and relates to an anti-African swine fever virus P22 protein antibody and an application thereof. Background Art

[0002] African swine fever is an acute, hemorrhagic, and highly contagious disease caused by the African swine fever virus infecting domestic pigs and wild boars. In August 2018, the first case of African swine fever in my country was reported in Shenyang, Liaoning Province, causing significant economic losses and social impact. Sequence analysis showed that the strain initially introduced into my country was a highly virulent genotype II strain. With the prevalence of African swine fever virus in my country, field detection and isolation of medium-virulence genotype II natural variants and low-virulence genotype I strains were detected and isolated. These strains have a long incubation period after infection, mild symptoms, and irregular detoxification, but they still have a strong ability to spread, making it more likely to spread over a large area and more difficult to eradicate.

[0003] ASFV is the only member of the genus Asfrinoviridae and the only arbovirus. The virus particle has a complex icosahedral symmetric structure, consisting of five layers: genome, nucleocapsid, inner membrane, capsid, and outer membrane. ASFV encodes 150-167 open reading frames (ORFs), encoding 68 structural proteins and more than 100 non-structural proteins.

[0004] The ASFV P22 protein is encoded by the KP177R gene. The ORF is located at the left end of the genome. The molecular weight is 22KDa. It is a transmembrane structural protein that is induced to express early after viral infection and is located in the inner membrane of the virus particle. The N-terminus of the P22 protein contains a hydrophobic region with a characteristic signal peptide. The exact function of the P22 protein is still unclear. High-throughput methods and proteomic analysis have shown that the P22 protein interacts with cellular proteins related to biological processes such as cell structure, signal transduction and cell adhesion, and contributes to phagocytosis. Studies have reported that the P22 protein expressed by baculovirus has good immunogenicity and can induce specific P22 antibodies after immunization of pigs. Therefore, it can be used as an antigen for serological detection. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a hybridoma cell, wherein the hybridoma cell is a hybridoma cell having a microbial deposit number of CCTCC NO: C2024215; or

[0006] A subculture cell of a hybridoma cell with a microbial deposit number of CCTCC NO: C2024215;

[0007] The monoclonal antibodies secreted by the passaged cells retain specific binding activity to the P22 protein of African swine fever virus.

[0008] A second aspect of the present invention is a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10;

[0009] P1: Monoclonal antibody

[0010] The monoclonal antibody retains specific binding activity to the African swine fever virus P22 protein;

[0011] The monoclonal antibody comprises a monoclonal antibody heavy chain and a monoclonal antibody light chain;

[0012] The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0013] The monoclonal antibody light chain includes a light chain CDR1, a light chain CDR2 and a light chain CDR3;

[0014] The heavy chain CDR1 protein sequence is shown in SEQ ID NO.4;

[0015] The heavy chain CDR2 protein sequence is shown in SEQ ID NO.6;

[0016] The heavy chain CDR3 protein sequence is shown in SEQ ID NO.8;

[0017] The light chain CDR1 protein sequence is shown in SEQ ID NO.10;

[0018] The light chain CDR2 protein sequence is shown in SEQ ID NO.12;

[0019] The light chain CDR3 protein sequence is shown in SEQ ID NO.14;

[0020] P2: Combination of monoclonal antibody heavy chain and monoclonal antibody light chain

[0021] The monoclonal antibody heavy chain and monoclonal antibody light chain combination maintains specific binding activity to African swine fever virus P22 protein;

[0022] The monoclonal antibody heavy chain includes the amino acid sequence of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and a functional protein fragment or an inert protein fragment;

[0023] The monoclonal antibody light chain includes the amino acid sequence of light chain CDR1, light chain CDR2, light chain CDR3 and a functional protein fragment or an inert protein fragment;

[0024] The heavy chain CDR1 protein sequence is shown in SEQ ID NO.4;

[0025] The heavy chain CDR2 protein sequence is shown in SEQ ID NO.6;

[0026] The heavy chain CDR3 protein sequence is shown in SEQ ID NO.8;

[0027] The light chain CDR1 protein sequence is shown in SEQ ID NO.10;

[0028] The light chain CDR2 protein sequence is shown in SEQ ID NO.12;

[0029] The light chain CDR3 protein sequence is shown in SEQ ID NO.14;

[0030] P3: Antibody derivatives

[0031] The antibody derivative retains specific binding activity to the African swine fever virus P22 protein;

[0032] The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3;

[0033] The heavy chain CDR1 protein sequence is shown in SEQ ID NO.4;

[0034] The heavy chain CDR2 protein sequence is shown in SEQ ID NO.6;

[0035] The heavy chain CDR3 protein sequence is shown in SEQ ID NO.8;

[0036] The light chain CDR1 protein sequence is shown in SEQ ID NO.10;

[0037] The light chain CDR2 protein sequence is shown in SEQ ID NO.12;

[0038] The light chain CDR3 protein sequence is shown in SEQ ID NO.14;

[0039] The antibody derivative is in the form selected from the group consisting of: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, porcine antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies;

[0040] P4: RNA combination

[0041] The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA;

[0042] The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or P2;

[0043] The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or P2;

[0044] P5: Genetic combination

[0045] The coding sequence of the gene combination can encode the monoclonal antibody heavy chain described in P1 or P2 and the monoclonal antibody light chain described in P1 or P2;

[0046] P6: Gene expression cassette combination

[0047] The gene expression product in the gene expression cassette combination is the RNA combination described in P4;

[0048] P7: Genetic Engineering Vector

[0049] The genetic engineering vector contains the gene expression cassette described in P6;

[0050] The monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in one or two vectors;

[0051] P8: Cells

[0052] The cell contains the genetic engineering vector described in P7;

[0053] The coding RNA in the gene expression cassette of the genetic engineering vector is constitutively expressed or artificially induced;

[0054] When the monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells;

[0055] P9: Composition

[0056] The composition contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8; and

[0057] P10: Test kit

[0058] The kit contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8.

[0059] In some embodiments, it is selected from any one of the following C1, C2, C3, C4 or a combination thereof:

[0060] C1: In P1, the monoclonal antibody is the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0061] C2: In P2, the functional protein fragment is a tag peptide and / or a signal peptide used for separating and purifying the protein;

[0062] C3: The African swine fever virus P22 protein sequence is shown in SEQ ID NO.2;

[0063] C4: The enzyme-labeled antibody is an antibody labeled with horseradish peroxidase.

[0064] The third aspect of the present invention provides the use of the hybridoma cell described in the first aspect of the present invention or the biological material described in the second aspect of the present invention in the preparation of a preparation for identifying, detecting or identifying African swine fever virus P22 protein, African swine fever virus subviral particles containing African swine fever virus P22 protein, or African swine fever virus particles.

[0065] A fourth aspect of the present invention provides a kit, which is the first kit or the second kit, and which contains a solid surface, and the solid surface is coated with an African swine fever virus P22 protein or a fusion protein containing an African swine fever virus P22 protein sequence;

[0066] The first kit also contains a monoclonal antibody 1 connected to a first marker or a combination of a monoclonal antibody heavy chain and a monoclonal antibody light chain connected to a first marker;

[0067] The monoclonal antibody 1 maintains specific binding activity to the African swine fever virus P22 protein;

[0068] The monoclonal antibody 1 comprises a monoclonal antibody heavy chain 1 and a monoclonal antibody light chain 1;

[0069] The monoclonal antibody heavy chain 1 includes a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2 and a first monoclonal antibody heavy chain CDR3;

[0070] The monoclonal antibody light chain 1 includes a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2 and a first monoclonal antibody light chain CDR3;

[0071] The heavy chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.4;

[0072] The heavy chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.6;

[0073] The heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.8;

[0074] The light chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.10;

[0075] The light chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.12;

[0076] The light chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.14;

[0077] The monoclonal antibody heavy chain and monoclonal antibody light chain combination maintains specific binding activity to African swine fever virus P22 protein;

[0078] The monoclonal antibody heavy chain and monoclonal antibody light chain combination comprises monoclonal antibody heavy chain 2 and monoclonal antibody light chain 2;

[0079] The monoclonal antibody heavy chain 2 includes the amino acid sequence of the second monoclonal antibody heavy chain CDR1, the second monoclonal antibody heavy chain CDR2, the second monoclonal antibody heavy chain CDR3 and a functional protein fragment or an inert protein fragment;

[0080] The monoclonal antibody light chain 2 includes the amino acid sequence of the second monoclonal antibody light chain CDR1, the second monoclonal antibody light chain CDR2, the second monoclonal antibody light chain CDR3 and a functional protein fragment or an inert protein fragment;

[0081] The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.4;

[0082] The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.6;

[0083] The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.8;

[0084] The light chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.10;

[0085] The light chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.12;

[0086] The light chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.14;

[0087] The second kit also contains monoclonal antibody 2 and a mouse IgG specific binding substance connected with a second marker;

[0088] The monoclonal antibody 2 is the monoclonal antibody secreted by the hybridoma cell described in the first aspect of the present invention.

[0089] In some embodiments, any one or a combination thereof is selected from the following D1, D2, D3, D4, D5:

[0090] D1: the monoclonal antibody 1 is the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0091] D2: the solid surface is the inner surface of the well of the microplate;

[0092] D3: The P22 protein sequence of the African swine fever virus is shown in SEQ ID NO.2;

[0093] D4: The first label is horseradish peroxidase or a fluorescent molecule;

[0094] D5: The second label is horseradish peroxidase or a fluorescent molecule.

[0095] In some embodiments, the kit further comprises any one or more combinations of the following materials:

[0096] Serum positive for African swine fever virus antibody;

[0097] Serum negative for African swine fever virus antibodies;

[0098] TMB substrate colorimetric solution;

[0099] PBS solution;

[0100] PBST solution;

[0101] Skim milk solution;

[0102] Aqueous sulfuric acid.

[0103] The fifth aspect of the present invention provides a method for detecting African swine fever virus antibodies for non-diagnostic purposes, wherein the method is method 1 or method 2;

[0104] The method 1 comprises the following steps:

[0105] P1: coating a solid surface with African swine fever virus P22 protein or a fusion protein containing the African swine fever virus P22 protein sequence to obtain a coated solid surface;

[0106] P2: adding the sample to be tested to the coated solid surface for incubation to obtain a solid surface for the first incubation;

[0107] P3: adding the monoclonal antibody 1 connected to the first marker or the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain connected to the first marker to the solid phase surface of the first incubation for incubation to obtain a solid phase surface of the second incubation;

[0108] The monoclonal antibody 1 maintains specific binding activity to the African swine fever virus P22 protein;

[0109] The monoclonal antibody 1 comprises a monoclonal antibody heavy chain 1 and a monoclonal antibody light chain 1;

[0110] The monoclonal antibody heavy chain 1 includes a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2 and a first monoclonal antibody heavy chain CDR3;

[0111] The monoclonal antibody light chain 1 includes a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2 and a first monoclonal antibody light chain CDR3;

[0112] The heavy chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.4;

[0113] The heavy chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.6;

[0114] The heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.8;

[0115] The light chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.10;

[0116] The light chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.12;

[0117] The light chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.14;

[0118] The monoclonal antibody heavy chain and monoclonal antibody light chain combination maintains specific binding activity to African swine fever virus P22 protein;

[0119] The monoclonal antibody heavy chain and monoclonal antibody light chain combination comprises monoclonal antibody heavy chain 2 and monoclonal antibody light chain 2;

[0120] The monoclonal antibody heavy chain 2 includes the amino acid sequence of the second monoclonal antibody heavy chain CDR1, the second monoclonal antibody heavy chain CDR2, the second monoclonal antibody heavy chain CDR3 and a functional protein fragment or an inert protein fragment;

[0121] The monoclonal antibody light chain 2 includes the amino acid sequence of the second monoclonal antibody light chain CDR1, the second monoclonal antibody light chain CDR2, the second monoclonal antibody light chain CDR3 and a functional protein fragment or an inert protein fragment;

[0122] The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.4;

[0123] The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.6;

[0124] The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.8;

[0125] The light chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.10;

[0126] The light chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.12;

[0127] The light chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.14;

[0128] P4: Characterizing the first marker indirectly bound to the solid surface of the second incubation, and determining whether there is African swine fever virus antibody in the sample to be tested according to the presence or absence of the first marker;

[0129] The method 2 comprises the following steps:

[0130] S1: coating a solid surface with African swine fever virus P22 protein or a fusion protein containing the African swine fever virus P22 protein sequence to obtain a coated solid surface;

[0131] S2: adding the sample to be tested to the coated solid surface for incubation to obtain a solid surface for the first incubation;

[0132] S3: adding monoclonal antibody 2 to the solid phase surface of the first incubation for incubation to obtain a solid phase surface of the second incubation;

[0133] The monoclonal antibody 2 is the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0134] S4: adding a mouse IgG specific binding substance connected with a second marker to the solid phase surface incubated for the second time to obtain a solid phase surface incubated for the third time;

[0135] S5: Characterize the second marker indirectly bound to the solid surface of the third incubation, and determine whether African swine fever virus antibodies exist in the sample to be tested based on the presence or absence of the second marker.

[0136] In some embodiments, it is selected from any one of the following E1, E2, E3, E4, E5 or a combination thereof:

[0137] E1: the monoclonal antibody 1 is the monoclonal antibody secreted by the hybridoma cell according to the first aspect of the present invention;

[0138] E2: The solid surface is the inner surface of the well of the microplate;

[0139] E3: The African swine fever virus P22 protein sequence is shown in SEQ ID NO.2;

[0140] E4: The sample to be tested is a sample in a solution state;

[0141] E5: The mouse IgG specific binder is an anti-mouse IgG antibody.

[0142] In some embodiments, it is selected from any one of the following F1, F2, F3 or a combination thereof:

[0143] F1: After any one of steps P1, P2, P3, S1, S2, S3 and S4 is completed, the solid phase surface is washed with PBS or PBST;

[0144] F2: The first marker is horseradish peroxidase, and the first marker is characterized by TMB substrate colorimetric solution, and OD is measured 450nm The value is used to determine whether the sample to be tested contains antibodies against the African swine fever virus P22 protein;

[0145] F3: The second marker is horseradish peroxidase, and TMB substrate colorimetric solution is used to characterize the second marker and measure OD 450nm The value is used to determine whether the sample to be tested contains antibodies against African swine fever virus P22 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0146] Figure 1 The blocking effect of African swine fever virus-positive serum on monoclonal antibody 2F8 is shown.

[0147] Figure 2 The results of enzyme-labeled antibody dilution optimization are shown.

[0148] Figure 3 Serum dilution optimization results are shown.

[0149] Figure 4The results of determination of the cut-off value by bELISA based on the enzyme-labeled 2F8 are shown.

[0150] Figure 5 A comparison chart of antibody levels detected by P72-iELISA and P22-bELISA is shown.

[0151] Figure 6 P22-b ELISA specificity test results are shown. DETAILED DESCRIPTION

[0152] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0153] Materials and instruments not described in the present invention are conventional materials and instruments in the art, operation details not described in the present invention are conventional operations in the art, the software used in the present invention is operated by conventional methods with reference to the instructions of the software provider, and the kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0154] The nucleic acid sequences shown in the present invention are all written from left to right in the direction of 5' to 3', and the proteins are written from left to right in the direction of N-terminus to C-terminus.

[0155] Application scenarios for detecting African swine fever virus for non-diagnostic purposes (not the process of identifying, studying and determining the cause or status of lesions in living humans or animals) include but are not limited to the following:

[0156] (1) Inoculating non-susceptible animals with African swine fever virus in order to study certain biological characteristics of the African swine fever virus through the method of detecting African swine fever virus antibodies for non-diagnostic purposes of the present invention.

[0157] (2) Pigs were inoculated with African swine fever virus P22 protein, and the immunological properties of the P22 protein were studied using the method of the present invention for detecting African swine fever virus antibodies for non-diagnostic purposes.

[0158] (3) The method for detecting African swine fever virus antibodies for non-diagnostic purposes of the present invention is used to detect the hybridoma cell culture fluid to obtain or identify a monoclonal antibody that binds to the same African swine fever virus P22 protein epitope as the antibody secreted by the hybridoma cell with the microbial preservation number of CCTCC NO: C2024215.

[0159] Example 1: Preparation and identification of monoclonal antibodies

[0160] (I) Preparation of African swine fever virus P22 protein

[0161] The present invention uses the KP177R gene of the African swine fever virus HLJ / 18 strain (GenBank No. MK333180.1, the full name of which is Pig / HLJ / 2018), and its coding sequence is as follows (SEQ ID NO.1):

[0162] ATGTTTAATATTAAAATGACAATTTCTACATTGCTTATTGCTCTTATTATACTACTTATTATTATTTTAGTAGTGTTTTTATACTATAAGAAACAACAACCACCGAAAAAGGTCTGTAAAGTAGATAAAGATT GTGGTAGTGGAGAGCATTGTGTTCGTGGATCATGTAGCTCATTGAGCTGCTTAGATGCCGTAAAAATGGACAAACGAAATTATTAAGATAGATTCTAAGATTTCCTCATGCGAATTCACTCCCAATTTTTACCGT TTTACGGATACTGCTGCTGATGAGCAGCAAGAATTTGGAAAAACACGGCATCCTATAAAAATAACTCCATCTCCAAGTGAATCCCATAGCCCCCAAGAGGTGTGTGAAAAATATTGTTCATGGGGAACCGATG ACTGTACAGGTTGGGAATATGTTGGTGATGAAAAGGAGGGAACATGTTATGTATAATAATCCACATCACCCGGTTCTTAAATATGGTAAGGATCACATCATAGCCTTACCTAGAAATCATAAACATGCATAA

[0163] The P22 protein sequence encoded by the aforementioned KP177R gene is as follows (SEQ ID NO.2):

[0164] MFNIKMTISTLLIALIILLIIILVVFLYYKKQQPPKKVCKVDKDCGSGEHCVRGSCSSLSCLDAVKMDKRNIKIDSKISSCEFTPNFYRFTDTAADEQQEFGKTRHPIKITPSPSESHSPQEVCEKYCSWGTDDCTGWEYVGDEKEGTCYVYNNPHHPVLKYGKDHIIALPRNHKHA

[0165] The pMAL-C5x recombinant plasmid containing the aforementioned KP177R gene coding sequence was transformed into the Escherichia coli ER2523 strain to induce the expression of exogenous P22 protein, and the StrepⅡ tag protein was introduced through primer design. At the same time, the plasmid carried maltose binding protein (MBP) as a tag protein, and then the obtained protein was purified by the StrepⅡ purification system to obtain the purified P22 recombinant protein.

[0166] (II) Preparation of monoclonal antibody against P22 protein

[0167] The purified P22 protein solution (1 mg / ml, the solvent is sterile PBS (0.01 mol / L, pH 7.4)) was mixed and emulsified with an equal volume of complete Freund's adjuvant, and 6-8 week old female BALB / c mice were subcutaneously immunized, a total of 6 mice, and the immunization dose was 50 μg P22 protein / mouse. Then, the same dose of P22 protein solution was mixed and emulsified with an equal volume of incomplete Freund's adjuvant to prepare the immunogen, and two booster immunizations were performed, with an interval of 2 weeks between each immunization. The dose was the same each time. Ten days after the second booster immunization, blood was collected from each mouse, serum was separated, and antibody titer was determined using the indirect P22-ELISA method. The mouse with the highest antibody titer was selected and the purified P22 protein solution was used for the last booster immunization without adjuvant, with an immunization dose of 50 μg P22 protein / mouse. After euthanasia, spleen cells were taken and fused with SP2 / 0 myeloma cells. The fused hybridoma cells were cultured in HAT selection culture for 10 days. The purified P22 protein prepared in step (i) was used as the coating antigen, and the P22 antibody in the cell supernatant 10 days after fusion was detected by indirect ELISA. The antibody-positive wells were subcloned three times by limiting dilution method, and finally a positive hybridoma cell with a single genetic background that can secrete antibodies against P22 protein was obtained, which was named cell line 2F8, and the monoclonal antibody secreted by the hybridoma cell was called 2F8.

[0168] (III) Typing of P22 protein monoclonal antibody and gene sequence determination

[0169] The monoclonal antibody 2F8 was typed and identified using a mouse immunoglobulin typing kit (supplier: Southern Biotech, catalog number 5300-05), and it was found that the monoclonal antibody was a kappa light chain, IgG2b type.

[0170] The total RNA of hybridoma 2F8 was obtained by Trizol method, reverse transcribed into cDNA, and multiple primer pairs were designed according to the conserved sequence of mouse antibody gene. The cDNA was amplified by PCR, and the amplified product was sequenced. The antibody heavy chain coding sequence and light chain coding sequence were spliced ​​by combining the use of NCBI NucleotideBLAST, IMGT / V Quest program and NCBI IgBLAST tools. The key sequences are as follows:

[0171] Heavy chain CDR1 coding sequence (SEQ ID NO.3):

[0172] AGCGCATGGATGCAC

[0173] Heavy chain CDR1 protein sequence (SEQ ID NO.4):

[0174] SAWMH

[0175] Heavy chain CDR2 coding sequence (SEQ ID NO.5:

[0176] GAGATTCATGCAAATAGTGGTACTACTAACTACAATGAGAAGTTCA AGGGC

[0177] Heavy chain CDR2 protein sequence (SEQ ID NO.6):

[0178] EIHANSGTTNYNEKFKG

[0179] Heavy chain CDR3 coding sequence (SEQ ID NO.7):

[0180] TCCGCCCCATTTGCTTAC

[0181] Heavy chain CDR3 protein sequence (SEQ ID NO.8):

[0182] SAPFAY

[0183] Light chain CDR1 coding sequence (SEQ ID NO.9):

[0184] AAGGCCAGTCAGGCAGTGGGTACTAATGTAGCC

[0185] Light chain CDR1 protein sequence (SEQ ID NO.10):

[0186] KASQAVGTNVA

[0187] Light chain CDR2 coding sequence (SEQ ID NO.11):

[0188] TCGGCATCCTACGCATACAGT

[0189] Light chain CDR2 protein sequence (SEQ ID NO.12):

[0190] SASYAYS

[0191] Light chain CDR3 coding sequence (SEQ ID NO.13):

[0192] CAGGCATATAACAGCTATCCATTCACG

[0193] Light chain CDR3 protein sequence (SEQ ID NO.14):

[0194] QAYNSYPFT

[0195] The hybridoma cell line 2F8 prepared by the present invention is submitted to a patent procedure approved depository for deposit. The depository is China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCC NO: C2024215; the culture name is hybridoma cell line 2F8; the Chinese classification name is: hybridoma cell; the English classification name is: Hybridoma Cell; the deposit time is July 26, 2024; the identified survival time is July 31, 2024.

[0196] Example 2: Evaluation of the blocking effect of P22 monoclonal antibody 2F8 on African swine fever positive serum and preliminary establishment of bELISA method

[0197] In order to evaluate the application prospect of monoclonal antibody 2F8 as an ASFV antibody detection reagent, a bELISA method based on monoclonal antibody 2F8 (referred to as P22-bELISA) was established. Six known ASFV-positive pig sera (prepared by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) and four ASFV-negative pig sera (prepared by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were selected to determine the performance of 2F8 and establish the bELISA method.

[0198] The P22-b ELISA test procedure is as follows:

[0199] 1. Coating: Dilute the African swine fever virus P22 protein antigen prepared in Example 1 into 4 μg / ml antigen dilution with antigen coating solution (carbonate buffer, pH 8.0), add to 96-well plate, 50 μl / well, and coat overnight at 4°C.

[0200] 2. Washing: Wash 5 times with 300 μl / well of PBST (phosphate buffered saline containing 0.05 v / v% Tween 20, pH 7.4, the same below), each time for 1 minute.

[0201] 3. Blocking: Block with 5 w / v% (5 g / 100 ml) skim milk (solvent: PBS), 200 μl / well, incubate at 37°C for 2 hours.

[0202] 4. Washing: Same as 2.

[0203] 5. Sample addition: dilute each serum with PBS at a volume ratio of 1:10, add to a 96-well plate, 50 μl / well, and incubate at 37°C for 2 hours.

[0204] 6. Washing: Same as 2.

[0205] 7. Add antibody: dilute 2F8 (1 mg / ml) with PBS at a volume ratio of 1:1000, 50 μl / well, and incubate at 37°C for 1 hour.

[0206] 8. Washing: Same as 2.

[0207] 9. Enzyme-labeled antibody: Dilute the enzyme-labeled antibody (horseradish peroxidase-labeled goat anti-mouse IgG antibody, concentration 0.4 mg / ml, purchased from OriGene, catalog number ZB-5305) at a volume ratio of 1:10,000 with PBS, 50 μl / well, and incubate at 37°C for 45 minutes.

[0208] 10. Washing: Same as 2.

[0209] 11. Color development: Add 50 μl TMB substrate color development solution (InnoReagents, TMB-S-004) to each well for color development and react at 37°C in the dark for 10 minutes.

[0210] 12. Stop: Add 50 μl stop solution (2M H2SO4) to each well.

[0211] 13. Reading: Measure OD within 5 minutes of stopping 450nm value.

[0212] According to the following formula, the blocking rate % = [(negative control average OD 450nm - Sample OD 450nm ) / mean OD of negative control 450nm ]×100.

[0213] bELISA blocking rate results refer to Figure 1 , in the figure, the PI values ​​of ASFV-positive pig sera (n=6) and negative sera (n=4) are shown as points on two vertical axes.

[0214] It can be seen that 2F8 can be blocked by ASFV positive pig serum and shows a high blocking rate. Next, the present invention further evaluated the P22 antibody 2F8 and used it to establish a monoclonal antibody-based bELISA method for ASFV antibody detection.

[0215] Example 3: Optimization of P22-b ELISA reaction conditions

[0216] The optimal dilutions of HRP-labeled 2F8 and serum samples were determined by systematic checkerboard titration.

[0217] (I) Optimization of enzyme-labeled antibody concentration

[0218] The affinity purified 2F8 was labeled with horseradish peroxidase to obtain horseradish peroxidase labeled monoclonal antibody 2F8, codenamed 2F8-HRP (concentration 1 mg / ml, solvent PBS), and 2F8-HRP was diluted with antibody diluent (InnoReagents, HRP-SD-001) at a volume ratio of 1:10000, 1:20000, 1:30000, 1:40000, 1:50000, 1:60000, 1:70000, 1:80000, 1:90000, and 1:100000. One of the 6 positive sera in Example 2 (diluted with PBS at a volume ratio of 1:10) and one of the 4 negative sera in Example 2 (diluted with PBS at a volume ratio of 1:10) were selected respectively. Step 7, step 8, and step 9 were combined into one step, specifically: for each of the aforementioned 2F8-HRP dilutions, 50 μl / well, incubated at 37° C. for 45 minutes, and other operating steps and parameters were the same as in Example 2, and a P22-b ELISA test was performed.

[0219] The experimental results can be found in Figure 2 It can be seen that the OD of each test well of the positive serum is close to 0, and complete blocking has occurred. When the dilution of 2F8-HRP antibody is 1:50000, the OD of the negative serum is 450nm The value was about 2.0, and the dilution of the enzyme-labeled antibody was determined to be 1:50,000.

[0220] (II) Optimization of serum dilution

[0221] In order to optimize the dilution of pig serum, in this step, the aforementioned 6 known ASFV-positive and 4 known ASFV-negative pig sera were diluted with PBS at 1:2, 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640 times, a total of 9 dilutions, and HRP-labeled 2F8 (2F8-HRP) selected a fixed dilution (1:50000). The other operating steps and parameters were the same as step (a) of Example 3, and the P22-b ELISA test was performed.

[0222] Blocking rate results can be found in Figure 3 , it can be seen that the serum dilution of 1:2 is the best.

[0223] (III) Optimization of incubation time

[0224] The serum sample and HRP monoclonal antibody (2F8-HRP) were added and the incubation time at 37°C was set to 30 min, 45 min, and 60 min, and the other operation steps and parameters were the same as step (ii) of Example 3, and the P22-b ELISA test was performed. The results showed that the 30 min time had the best effect.

[0225] The subsequent test examples are all conducted based on the conditions optimized in this step.

[0226] Example 4: Determination of P22-b ELISA cut-off value

[0227] A total of 350 pig serum samples were used, and 296 were determined to be ASFV antibody negative and 54 were determined to be ASFV antibody positive by commercial test kits. The P22-bELISA test established and optimized by Example 3 of the present invention was used to calculate the average blocking rate and standard deviation (SD) of the 296 negative samples, and the average value + 3SD was used as the judgment standard of the detection method, that is, the cut-off value. The results showed that the average blocking rate of negative samples was 15.2%, the SD value was 9.7%, and the calculated cut-off value was 44.4%. ROC curve analysis showed that the sensitivity was 100% and the specificity was 99.66% ( Figure 4 ).

[0228] Example 5: Evaluation of the ability of the established bELISA to detect antibodies in pigs infected with the Chinese medium-virulence gene type II variant and low-virulence gene type I ASFV strain

[0229] Early research by the National African Swine Fever Laboratory found that low-lethal genotype II African swine fever variants and genotype I African swine fever virus strains have appeared in my country. These virus strains cause chronic persistent infection, and infected pigs continue to carry and excrete toxins at a low level. Therefore, in order to verify whether the established method can be used to detect the serum of pigs infected with these viruses, 4 experimental pigs were infected with ASFV genotype II isolate Pig / HLJ / HRB1 / 20 (GenBank No. MW656282.1) by intramuscular injection, with an inoculation dose of 10 6 TCID 50 The other 6 experimental pigs were infected with ASFV genotype I isolate SD / DY-I / 21 (GenBank No. MZ945537.1) by intramuscular injection at a dose of 10 6 TCID 50 / Head. Blood was collected and serum was separated every 1 day before and after vaccination for 27 days, and the P22-bELISA established and optimized in Example 3 of the present invention was used for detection, and the commercial African swine fever virus P72 protein indirect ELISA (P72-iELISA) kit was used for detection. The results showed that one pig's P22 antibody turned positive on the 7th day after infection with the genotype II strain, and all 4 pigs' P22 antibodies turned positive on the 9th day; all 6 pigs' P22 antibodies turned positive on the 9th day after infection with the genotype I strain; the P72-iELISA test results were basically consistent with the P22-bELISA, and both genotypes began to turn positive in P72 antibodies on the 9th day after infection, but the P72 antibodies of the genotype II strain infection were suspected on the 9th day, and 3 pigs were confirmed positive until the 11th day, and even one pig's P72 antibody turned positive on the 13th day, and one pig's P72 antibody was not detected positive on the 9th day after infection with the genotype I strain, until the 13th day P72 antibody turned positive. This indicates that the antibody conversion time of P22-bELISA is earlier than that of P72-iELISA ( Figure 5 ).

[0230] Example 6: P22-b ELISA specificity test

[0231] The bELISA based on P22-2F8 optimized in Example 3 of the present invention was specifically evaluated using positive pig sera against several other major pig viruses. Using the conditions optimized in Example 3, CSFV (classical swine fever virus) positive pig serum (blocking ELISA antibody titer 1:64), PRRSV (porcine reproductive and respiratory syndrome virus) positive pig serum (indirect ELISA antibody titer 1:100), PRV (pseudorabies virus) positive pig serum (blocking ELISA antibody titer 1:128), PCV2 (porcine circovirus type 2) positive pig serum (indirect ELISA antibody titer 1:3200), PEDV (porcine epidemic diarrhea virus) positive pig serum (neutralizing antibody titer 1:16), TGEV (transmissible gastroenteritis virus) positive pig serum (neutralizing antibody titer 1:32), ASFV (African swine fever virus) positive pig serum (IFA antibody titer 1:6400) were used as sera to be tested, and the test conditions were parallel. For test results, see Figure 6 It can be seen that the positive sera of 6 major pig pathogens such as CSFV have no blocking effect, and the PI value is 9.1% to 21.2%, which shows negative. The ASFV positive pig serum has an obvious blocking effect, and the PI value is 97.7%.

[0232] It can be seen that the P22-b ELISA constructed in the present invention has good specificity for detecting African swine fever virus antiserum and meets the requirements for clinical use.

[0233] Example 7: Analysis of the coincidence rate between P22-bELISA and P72-iELISA

[0234] 878 serum samples (including local clinical serum samples and experimental infected pig serum) were tested by the P22-bELISA method established and optimized in Example 3 of the present invention and the commercial P72-iELISA. The results showed that the sensitivity of the P22-bELISA method of the present invention was 100.00%, the specificity was 99.30%, and the consistency rate with the P72-iELISA method was 98.18% (see Table 1).

[0235] Table 1. P22-bELISA and P72-iELISA Coincidence Test P72-iELISA Detection

[0236]

[0237] a: the number of samples with positive results in both P72-iELISA and P22-bELISA tests; b: the number of samples with positive results in P72-iELISA and negative results in P22-bELISA tests; c: the number of samples with negative results in P72-iELISA and positive results in P22-bELISA tests; d: the number of samples with negative results in both P72-iELISA and P22-bELISA tests; e: sensitivity % = a / (a+b)×100; f: specificity % = d / (c+d)×100; g: conformity % = (a+d) / (a+b+c+d)×100.

[0238] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A hybridoma cell, wherein the hybridoma cell is a hybridoma cell with a microbial deposit number of CCTCC NO: C2024215.

2. A biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10; P1: Monoclonal antibody The monoclonal antibody retains specific binding activity to the African swine fever virus P22 protein; The monoclonal antibody comprises a monoclonal antibody heavy chain and a monoclonal antibody light chain; The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The monoclonal antibody light chain includes a light chain CDR1, a light chain CDR2 and a light chain CDR3; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.4; The heavy chain CDR2 protein sequence is shown in SEQ ID NO.6; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.8; The light chain CDR1 protein sequence is shown in SEQ ID NO.10; The light chain CDR2 protein sequence is shown in SEQ ID NO.12; The light chain CDR3 protein sequence is shown in SEQ ID NO.14; P2: Combination of monoclonal antibody heavy chain and monoclonal antibody light chain The monoclonal antibody heavy chain and monoclonal antibody light chain combination maintains specific binding activity to African swine fever virus P22 protein; The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and a tag peptide and / or a signal peptide for separating and purifying the protein; The monoclonal antibody light chain includes a light chain CDR1, a light chain CDR2, a light chain CDR3 and a tag peptide and / or a signal peptide for separating and purifying the protein; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.4; The heavy chain CDR2 protein sequence is shown in SEQ ID NO.6; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.8; The light chain CDR1 protein sequence is shown in SEQ ID NO.10; The light chain CDR2 protein sequence is shown in SEQ ID NO.12; The light chain CDR3 protein sequence is shown in SEQ ID NO.14; P3: Antibody derivatives The antibody derivative retains specific binding activity to the African swine fever virus P22 protein; The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3; The heavy chain CDR1 protein sequence is shown in SEQ ID NO.4; The heavy chain CDR2 protein sequence is shown in SEQ ID NO.6; The heavy chain CDR3 protein sequence is shown in SEQ ID NO.8; The light chain CDR1 protein sequence is shown in SEQ ID NO.10; The light chain CDR2 protein sequence is shown in SEQ ID NO.12; The light chain CDR3 protein sequence is shown in SEQ ID NO.14; The antibody derivative is in the form selected from the group consisting of: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, porcine antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies; P4: RNA combination The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA; The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or P2; The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or P2; P5: Genetic combination The coding sequence of the gene combination can encode the monoclonal antibody heavy chain described in P1 or P2 and the monoclonal antibody light chain described in P1 or P2; P6: Gene expression cassette combination The gene expression product in the gene expression cassette combination is the RNA combination described in P4; P7: Genetic Engineering Vector The genetic engineering vector contains the gene expression cassette described in P6; The monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in one or two vectors; P8: Cells The cell contains the genetic engineering vector described in P7; The coding gene in the gene expression cassette of the genetic engineering vector is constitutively expressed or artificially induced; When the monoclonal antibody heavy chain RNA and the monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells; P9: Composition The composition contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8; and P10: Test kit The kit contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8; The African swine fever virus P22 protein sequence is shown in SEQ ID NO.

2.

3. The biomaterial according to claim 2, characterized in that Selected from one or a combination of the following C1 and C4: C1: In P1, the monoclonal antibody is the monoclonal antibody secreted by the hybridoma cell of claim 1; C4: The enzyme-labeled antibody is an antibody labeled with horseradish peroxidase.

4. Use of the hybridoma cell according to claim 1 or the biological material according to claim 2 or 3 in preparing a preparation for identifying, detecting or identifying African swine fever virus P22 protein, African swine fever virus subviral particles containing African swine fever virus P22 protein, or African swine fever virus particles; the African swine fever virus P22 protein sequence is shown in SEQ ID NO.

2.

5. A kit, the kit being the first kit or the second kit, the kit comprising a solid surface, the solid surface being coated with African swine fever virus P22 protein or a fusion protein containing an African swine fever virus P22 protein sequence; the African swine fever virus P22 protein sequence is shown as SEQ ID NO.2; The first kit also contains a monoclonal antibody 1 connected to a first marker or a combination of a monoclonal antibody heavy chain and a monoclonal antibody light chain connected to a first marker; The monoclonal antibody 1 maintains specific binding activity to the African swine fever virus P22 protein; The monoclonal antibody 1 comprises a monoclonal antibody heavy chain 1 and a monoclonal antibody light chain 1; The monoclonal antibody heavy chain 1 includes a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2 and a first monoclonal antibody heavy chain CDR3; The monoclonal antibody light chain 1 includes a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2 and a first monoclonal antibody light chain CDR3; The heavy chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.4; The heavy chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.6; The heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.8; The light chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.10; The light chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.12; The light chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.14; The monoclonal antibody heavy chain and monoclonal antibody light chain combination maintains specific binding activity to African swine fever virus P22 protein; The monoclonal antibody heavy chain and monoclonal antibody light chain combination comprises monoclonal antibody heavy chain 2 and monoclonal antibody light chain 2; The monoclonal antibody heavy chain 2 includes a second monoclonal antibody heavy chain CDR1, a second monoclonal antibody heavy chain CDR2, a second monoclonal antibody heavy chain CDR3 and a tag peptide and / or a signal peptide for separating and purifying the protein; The monoclonal antibody light chain 2 comprises a second monoclonal antibody light chain CDR1, a second monoclonal antibody light chain CDR2, a second monoclonal antibody light chain CDR3 and a tag peptide and / or a signal peptide for separating and purifying the protein; The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.4; The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.6; The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.8; The light chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.10; The light chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.12; The light chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.14; The second kit also contains monoclonal antibody 2 and a mouse IgG specific binding substance connected with a second marker; The monoclonal antibody 2 is the monoclonal antibody secreted by the hybridoma cell according to claim 1.

6. The kit according to claim 5, characterized in that Selected from any one of the following D1, D2, D4, D5 or a combination thereof: D1: the monoclonal antibody 1 is the monoclonal antibody secreted by the hybridoma cell according to claim 1; D2: the solid surface is the inner surface of the well of the microplate; D4: The first label is horseradish peroxidase or a fluorescent molecule; D5: The second label is horseradish peroxidase or a fluorescent molecule.

7. The kit according to claim 5 or 6, characterized in that The kit also contains any one or more combinations of the following materials: Serum positive for African swine fever virus antibody; African swine fever virus antibody-negative serum; TMB substrate colorimetric solution; PBS solution; PBST solution; Skim milk solution; Aqueous sulfuric acid.

8. A method for detecting African swine fever virus antibodies for non-diagnostic purposes, the method being method 1 or method 2; The method 1 comprises the following steps: P1: coating a solid surface with African swine fever virus P22 protein or a fusion protein containing the African swine fever virus P22 protein sequence to obtain a coated solid surface; P2: adding the sample to be tested to the coated solid surface for incubation to obtain a solid surface for the first incubation; P3: adding the monoclonal antibody 1 connected to the first marker or the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain connected to the first marker to the solid phase surface of the first incubation for incubation to obtain a solid phase surface of the second incubation; The monoclonal antibody 1 maintains specific binding activity to the African swine fever virus P22 protein; The monoclonal antibody 1 comprises a monoclonal antibody heavy chain 1 and a monoclonal antibody light chain 1; The monoclonal antibody heavy chain 1 includes a first monoclonal antibody heavy chain CDR1, a first monoclonal antibody heavy chain CDR2 and a first monoclonal antibody heavy chain CDR3; The monoclonal antibody light chain 1 includes a first monoclonal antibody light chain CDR1, a first monoclonal antibody light chain CDR2 and a first monoclonal antibody light chain CDR3; The heavy chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.4; The heavy chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.6; The heavy chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.8; The light chain CDR1 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.10; The light chain CDR2 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.12; The light chain CDR3 protein sequence of the first monoclonal antibody is shown in SEQ ID NO.14; The monoclonal antibody heavy chain and monoclonal antibody light chain combination maintains specific binding activity to African swine fever virus P22 protein; The monoclonal antibody heavy chain and monoclonal antibody light chain combination comprises monoclonal antibody heavy chain 2 and monoclonal antibody light chain 2; The monoclonal antibody heavy chain 2 includes a second monoclonal antibody heavy chain CDR1, a second monoclonal antibody heavy chain CDR2, a second monoclonal antibody heavy chain CDR3 and a tag peptide and / or a signal peptide for separating and purifying the protein; The monoclonal antibody light chain 2 includes a second monoclonal antibody light chain CDR1, a second monoclonal antibody light chain CDR2, a second light chain CDR3 and a tag peptide and / or a signal peptide for separating and purifying the protein; The heavy chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.4; The heavy chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.6; The heavy chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.8; The light chain CDR1 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.10; The light chain CDR2 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.12; The light chain CDR3 protein sequence of the second monoclonal antibody is shown in SEQ ID NO.14; P4: Characterizing the first marker indirectly bound to the solid surface of the second incubation, and determining whether there is African swine fever virus antibody in the sample to be tested according to the presence or absence of the first marker; The method 2 comprises the following steps: S1: coating a solid surface with African swine fever virus P22 protein or a fusion protein containing the African swine fever virus P22 protein sequence to obtain a coated solid surface; S2: adding the sample to be tested to the coated solid surface for incubation to obtain a solid surface for the first incubation; S3: adding monoclonal antibody 2 to the solid phase surface of the first incubation for incubation to obtain a solid phase surface of the second incubation; The monoclonal antibody 2 is the monoclonal antibody secreted by the hybridoma cell according to claim 1; S4: adding a mouse IgG specific binding substance connected with a second marker to the solid phase surface incubated for the second time to obtain a solid phase surface incubated for the third time; S5: Characterize the second marker indirectly bound to the solid surface of the third incubation, and determine whether African swine fever virus antibodies exist in the sample to be tested based on the presence or absence of the second marker.

9. The method according to claim 8, characterized in that Selected from any one of the following E1, E2, E3, E4, E5 or a combination thereof: E1: the monoclonal antibody 1 is the monoclonal antibody secreted by the hybridoma cell according to claim 1; E2: The solid surface is the inner surface of the well of the microplate; E3: The African swine fever virus P22 protein sequence is shown in SEQ ID NO.2; E4: The sample to be tested is a sample in a solution state; E5: The mouse IgG specific binder is an anti-mouse IgG antibody.

10. The method according to claim 8 or 9, characterized in that Selected from any one of the following F1, F2, F3 or a combination thereof: F1: After any one of steps P1, P2, P3, S1, S2, S3 and S4 is completed, the solid phase surface is washed with PBS or PBST; F2: The first marker is horseradish peroxidase, and the first marker is characterized by TMB substrate colorimetric solution, and OD is measured 450nm The value is used to determine whether the sample to be tested contains antibodies against the African swine fever virus P22 protein; F3: The second marker is horseradish peroxidase, and TMB substrate colorimetric solution is used to characterize the second marker and measure OD 450nm The value is used to determine whether the sample to be tested contains antibodies against African swine fever virus P22 protein.

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