African swine fever virus monoclonal antibody and its application

Abstract

The present invention discloses a hybridoma cell, and its microbial deposit number is CCTCC NO: C202402. The present invention also discloses a monoclonal antibody secreted by the hybridoma cell. The present invention also discloses a blocking ELISA method and a kit based on the monoclonal antibody and African swine fever virus p72 protein labeled with horseradish peroxidase. The ELISA method can effectively detect African swine fever antibodies, has good sensitivity and specificity, and has clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products and relates to an African swine fever virus monoclonal 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. The strain that initially circulated in my country was a highly virulent genotype II strain. In recent years, studies have reported the emergence of moderately virulent genotype II natural variants and low-virulence genotype I strains. These strains have a long incubation period after infection, mild symptoms, and irregular toxin excretion, 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 Asfivirus in the family Asfiviridae 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 approximately 150-167 open reading frames (ORFs), encoding 68 structural proteins and more than 100 non-structural proteins. p72, encoded by the B646L gene, is the major viral capsid protein, accounting for approximately 31-33% of the mass of the virus particle. The structure of p72 was resolved by cryo-electron microscopy studies, showing that the protein has an N-terminal base, a central jellyroll (JR) fold domain, a crown generated by insertion into the JR chain, and three p72 molecules assembled into a trimeric spike protruding outside the capsid. The trimeric p72 consists of four protruding ring structures, ER1, ER2, ER3, and ER4, which are considered to be key protective epitopes. Although some studies have generated and identified p72 monoclonal antibodies, information about the key epitopes of p72 is still limited. In addition, the p72 protein sequence is highly conserved among different ASFV strains. It is one of the most antigenic and immunogenic ASFV proteins and is the preferred target protein for ASF serological diagnosis and vaccine development.

[0004] Although low-virulence ASFV infection causes atypical symptoms, it can induce a strong humoral immune response, and antibodies can generally be detected 7 days after infection. Since low-virulence infection excretion is irregular, nucleic acid testing is prone to miss detection, so it is particularly important to cooperate with antibody testing for disease diagnosis. Summary of the invention

[0005] The present invention prepares a monoclonal antibody 2F11 against the p72 protein of African swine fever virus, and studies its characteristics. Based on this, the present invention establishes a rapid, sensitive and specific blocking ELISA (bELISA) detection method based on 2F11, which can effectively detect genotype I ASFV antibodies and genotype II ASFV antibodies. And further identified the linear B cell epitopes associated with 2F11. The present invention establishes a bELISA method based on monoclonal antibodies, which lays a foundation for further improving the detection level of ASFV antibodies.

[0006] The present invention screened and prepared p72-specific monoclonal antibodies, and established a bELISA method for detecting p72 antibodies. The method has high sensitivity, strong specificity, and good repeatability. The earliest time for the antibody conversion of laboratory infected pigs is 7 days after infection. The detection method provides technical support for the accurate and timely implementation of strict prevention and control measures for African swine fever in the early stage.

[0007] The first aspect of the present invention provides a hybridoma cell, wherein the microbial preservation number of the hybridoma cell is CCTCC NO: C202402.

[0008] The second aspect of the present invention provides a monoclonal antibody, which is the monoclonal antibody secreted by the hybridoma cell of the first aspect of the present invention or the monoclonal antibody secreted by the subculture cell of the hybridoma cell of the first aspect of the present invention.

[0009] The third aspect of the present invention provides a monoclonal antibody, which comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chain CDR1, a light chain CDR2 and a light chain CDR3;

[0010] The heavy chain CDR1 protein sequence is shown in SEQ ID NO.4;

[0011] The heavy chain CDR2 protein sequence is shown in SEQ ID NO.6;

[0012] The heavy chain CDR3 protein sequence is shown in SEQ ID NO.8;

[0013] The light chain CDR1 protein sequence is shown in SEQ ID NO.10;

[0014] The light chain CDR2 protein sequence is shown in SEQ ID NO.12;

[0015] The light chain CDR3 protein sequence is shown in EQ ID NO.14.

[0016] In some embodiments, the monoclonal antibody is a porcinized monoclonal antibody.

[0017] The fourth aspect of the present invention provides a monoclonal antibody gene combination, the monoclonal antibody gene combination comprises a heavy chain gene and a light chain gene, the coding sequence fragment of the heavy chain gene comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the coding sequence fragment of the light chain gene comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3;

[0018] The heavy chain CDR1 coding sequence is shown in SEQ ID NO.3;

[0019] The heavy chain CDR2 coding sequence is shown in SEQ ID NO.5;

[0020] The heavy chain CDR3 coding sequence is shown in SEQ ID NO.7;

[0021] The light chain CDR1 coding sequence is shown in SEQ ID NO.9;

[0022] The light chain CDR2 coding sequence is shown in SEQ ID NO.11;

[0023] The light chain CDR3 coding sequence is shown in SEQ ID NO.13.

[0024] In some embodiments, the monoclonal antibody is a porcinized monoclonal antibody.

[0025] The fifth aspect of the present invention provides an antibody variable region protein sequence combination, wherein the peptide segment of the antibody variable region protein sequence combination includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3;

[0026] The heavy chain CDR1 protein sequence is shown in SEQ ID NO.4;

[0027] The heavy chain CDR2 protein sequence is shown in SEQ ID NO.6;

[0028] The heavy chain CDR3 protein sequence is shown in SEQ ID NO.8;

[0029] The light chain CDR1 protein sequence is shown in SEQ ID NO.10;

[0030] The light chain CDR2 protein sequence is shown in SEQ ID NO.12;

[0031] The light chain CDR3 protein sequence is shown in EQ ID NO.14.

[0032] The sixth aspect of the present invention provides an antibody gene variable region nucleic acid sequence combination, wherein the gene fragment of the antibody gene variable region nucleic acid sequence combination includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 and light chain CDR3;

[0033] The heavy chain CDR1 coding sequence is shown in SEQ ID NO.3;

[0034] The heavy chain CDR2 coding sequence is shown in SEQ ID NO.5;

[0035] The heavy chain CDR3 coding sequence is shown in SEQ ID NO.7;

[0036] The light chain CDR1 coding sequence is shown in SEQ ID NO.9;

[0037] The light chain CDR2 coding sequence is shown in SEQ ID NO.11;

[0038] The light chain CDR3 coding sequence is shown in SEQ ID NO.13.

[0039] The seventh aspect of the present invention provides an antibody derivative, which retains specific binding activity to the African swine fever virus p72 protein, and the protein sequence portion of the antibody derivative contains the antibody variable region protein sequence combination described in the fifth aspect of the present invention; the form of the antibody derivative is selected from: 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] In some embodiments, the amino acid sequence of the African swine fever p72 protein is as shown in SEQ ID NO.2.

[0041] The eighth aspect of the present invention provides a fusion protein, which retains specific binding activity to the African swine fever virus p72 protein, and the amino acid sequence of the fusion protein contains a combination of the antibody variable region protein sequence described in the fifth aspect of the present invention and a functional protein fragment or an inert fragment.

[0042] In some embodiments, the amino acid sequence of the African swine fever p72 protein is as shown in SEQ ID NO.2.

[0043] In some embodiments, the functional protein fragment is a tag peptide used to separate and purify the protein.

[0044] The ninth aspect of the present invention provides a polypeptide, the amino acid sequence of which is shown from the mth to the nth positions of SEQ ID NO.2, wherein m is any natural number between 199 and 283, and n is any natural number between 288 and 332.

[0045] The tenth aspect of the present invention provides a fusion protein, the amino acid sequence of which contains the polypeptide described in the ninth aspect of the present invention and a functional protein fragment or an inert fragment.

[0046] In some embodiments, the functional protein fragment is a tag peptide used to separate and purify the protein.

[0047] The eleventh aspect of the present invention provides a gene expression cassette, the coding sequence in the gene expression cassette can encode the monoclonal antibody described in the second aspect of the present invention, the monoclonal antibody described in the third aspect of the present invention, the fusion protein described in the eighth aspect of the present invention, the polypeptide described in the ninth aspect of the present invention or the fusion protein described in the tenth aspect of the present invention.

[0048] The twelfth aspect of the present invention provides a genetically engineered expression vector, wherein the genetically engineered expression vector contains the gene expression cassette described in the eleventh aspect of the present invention.

[0049] The thirteenth aspect of the present invention provides a host cell, wherein the host cell contains the genetically engineered expression vector described in the twelfth aspect of the present invention, and the encoded protein in the expression cassette of the genetically engineered expression vector is constitutively expressed or artificially induced.

[0050] The fourteenth aspect of the present invention provides an ELISA kit, which contains a microplate, and the bottom of the wells of the microplate is coated with the polypeptide described in the ninth aspect of the present invention or the fusion protein described in the tenth aspect of the present invention.

[0051] In some embodiments, in the step of preparing the microplate, the concentration of the polypeptide described in the ninth aspect of the present invention or the fusion protein described in the tenth aspect of the present invention in the coating solution is 1-3 μg / ml, and the coating amount per well is 50-200 μl.

[0052] In some embodiments, the kit further comprises the monoclonal antibody of the second aspect of the invention labeled with horseradish peroxidase, the monoclonal antibody of the third aspect of the invention labeled with horseradish peroxidase, or the antibody derivative of the seventh aspect of the invention labeled with horseradish peroxidase.

[0053] In some embodiments, the kit also contains African swine fever virus-negative pig serum and / or African swine fever virus-positive pig serum.

[0054] The fifteenth aspect of the present invention provides a kit, which contains the hybridoma cell described in the first aspect of the present invention, the monoclonal antibody described in the second aspect of the present invention, the monoclonal antibody described in the third aspect of the present invention, the antibody derivative described in the seventh aspect of the present invention, the fusion protein described in the eighth aspect of the present invention, the polypeptide described in the ninth aspect of the present invention, the fusion protein described in the tenth aspect of the present invention, the genetically engineered expression vector described in the twelfth aspect of the present invention, or the host cell described in the thirteenth aspect of the present invention.

[0055] A sixteenth aspect of the present invention provides a method for identifying African swine fever virus for non-diagnostic purposes, the method comprising the following steps:

[0056] S1: Infect African swine fever virus susceptible cells with the sample to be tested to obtain infected cells;

[0057] S2: fixing the infected cells on a solid surface to obtain immobilized cells;

[0058] S3: adding the monoclonal antibody described in the second aspect of the present invention or the monoclonal antibody described in the third aspect of the present invention to the immobilized cells for incubation to obtain immobilized cells for the first incubation;

[0059] S4: adding the monoclonal antibody specific binding substance connected with the label to the fixed cells of the first incubation for incubation to obtain the fixed cells of the second incubation;

[0060] S5: Characterizing the marker indirectly bound to the fixed cells in the second incubation, and determining whether African swine fever virus exists in the sample to be tested based on the presence or absence of the marker.

[0061] In some embodiments, in step S1, the sample to be tested is pig serum.

[0062] In some embodiments, in step S1, the African swine fever virus susceptible cells are wild boar kidney cells or primary porcine alveolar macrophages with a microbial collection number of CCTCC NO: C2022258.

[0063] In some embodiments, in step S2, the solid surface is the inner surface of a well of a microplate.

[0064] In some embodiments, in step S4, the monoclonal antibody specific binding substance connected with a label is a secondary antibody against the monoclonal antibody of the second aspect of the present invention or the monoclonal antibody of the third aspect of the present invention labeled with a fluorescent molecule.

[0065] In some embodiments, in step S5, the fixed cells incubated for the second time are photographed using a fluorescence microscope, and whether African swine fever virus is present in the sample to be tested is determined based on the photographic results.

[0066] A seventeenth aspect of the present invention provides a method for identifying African swine fever virus antibodies for non-diagnostic purposes, the method comprising the following steps:

[0067] P1: coating a solid surface with African swine fever p72 protein, an African swine fever p72 protein fusion protein containing a tag peptide for separating and purifying the protein, the polypeptide described in the ninth aspect of the present invention, or the fusion protein described in the tenth aspect of the present invention to obtain a coated solid surface;

[0068] P2: adding the sample to be tested to the coated solid surface for incubation to obtain a solid surface for the first incubation;

[0069] P3: adding the monoclonal antibody of the second aspect of the present invention connected with a label, the monoclonal antibody of the third aspect of the present invention connected with a label, or the antibody derivative of the seventh aspect of the present invention connected with a label to the solid phase surface of the first incubation for incubation to obtain a solid phase surface of the second incubation;

[0070] P4: Characterize the label that is indirectly bound to the solid surface of the second incubation, and determine whether African swine fever virus antibodies are present in the sample to be tested based on the presence or absence of the label.

[0071] In some embodiments, in step P1, the amino acid sequence of the African swine fever p72 protein is as shown in SEQ ID NO.2.

[0072] In some embodiments, in step P1, the solid surface is the inner surface of a well of a microplate.

[0073] In some embodiments, in step P2, the sample to be tested is pig serum to be tested.

[0074] In some embodiments, in step P2, African swine fever virus-negative pig serum is added in parallel as a negative control; and / or African swine fever virus-positive pig serum is added in parallel as a positive control.

[0075] In some embodiments, in step P2, the African swine fever virus-negative pig serum is diluted with a PBS solution at a volume ratio of 1:1 to 1:10 before use, and / or the African swine fever virus-positive pig serum is diluted with a PBS solution at a volume ratio of 1:1 to 1:10 before use.

[0076] In some embodiments, in step P3, the label is horseradish peroxidase, and in step P4, TMB substrate colorimetric solution is added to the solid surface of the second incubation, and the OD value of the solid surface of the second incubation is measured. 450nm The value is calculated to determine whether African swine fever virus antibodies exist in the sample to be tested.

[0077] In some embodiments, in step P3, the label is horseradish peroxidase, and the solvent of the monoclonal antibody of the second aspect of the present invention connected with a label, the monoclonal antibody of the third aspect of the present invention connected with a label, or the antibody derivative of the seventh aspect of the present invention connected with a label is PBS solution with a concentration of 50-200 μg / L.

[0078] In some embodiments, the identification is performed using the ELISA kit described in the fourteenth aspect of the present invention.

[0079] The eighteenth aspect of the present invention provides the use of the polypeptide described in the ninth aspect of the present invention or the fusion protein described in the tenth aspect of the present invention in screening or characterizing African swine fever virus antibodies.

[0080] The nineteenth aspect of the present invention provides the hybridoma cell described in the first aspect of the present invention, the monoclonal antibody described in the second aspect of the present invention, the monoclonal antibody described in the third aspect of the present invention, the monoclonal antibody gene combination described in the fourth aspect of the present invention, the antibody variable region protein sequence combination described in the fifth aspect of the present invention, the antibody gene variable region nucleic acid sequence combination described in the sixth aspect of the present invention, the antibody derivative described in the seventh aspect of the present invention, the fusion protein described in the eighth aspect of the present invention, the polypeptide described in the ninth aspect of the present invention, the fusion protein described in the tenth aspect of the present invention, the gene expression cassette described in the eleventh aspect of the present invention, the genetically engineered expression vector described in the twelfth aspect of the present invention, or the host cell described in the thirteenth aspect of the present invention for preparing a preparation for detecting African swine fever antibodies or African swine fever antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The identification results of 2F11 are shown.

[0082] Figure 2 Shown are the optimization results of the p72-b ELISA method.

[0083] Figure 3 The detection sensitivity, positive judgment threshold and performance evaluation results of p72-b ELISA using different groups of pig sera are shown.

[0084] Figure 4 p72-b ELISA specific detection results are shown.

[0085] Figure 5 The results of WB identification of p72 truncated fusion protein are shown.

[0086] Figure 6 The sequence information and WB identification map of the linear epitope recognized by 2F11 are shown.

[0087] Figure 7 The results of the linear B cell epitope characterization analysis of the p72 protein are shown. DETAILED DESCRIPTION

[0088] 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.

[0089] 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.

[0090] 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.

[0091] One of the application scenarios of African swine fever virus antibody testing for non-diagnostic purposes is to study the cross-immunity between African swine fever virus and other viruses.

[0092] One of the application scenarios for identifying African swine fever virus for non-diagnostic purposes is to evaluate the pig farm environment in order to develop disinfection plans.

[0093] Strains, cells and biomaterials

[0094] (1) BK2258 cells

[0095] BK2258 cells are a type of boar kidney cell, prepared and preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0096] The preparation method of BK2258 cells is recorded in the application documents of the Chinese patent application with application number CN202211137583.3. The isolated and cultured BK2258 strain F20 cells were submitted to a patent procedure approved depository for preservation. The depository is the China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCCNO: C2022258; the culture name is wild boar kidney cell BK2258; the Chinese classification name is: wild boar kidney cell; the English classification name is: Boar kidney cell; the preservation time is August 10, 2022; the identified survival time is August 17, 2022.

[0097] (2) Primary porcine alveolar macrophages

[0098] Primary porcine alveolar macrophages (PAMs) were obtained from 30-50 day old healthy SPF pigs and cultured in 1640 medium containing 10% (v / v) FBS in a 37°C, 5% CO2 incubator. They were prepared and used by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0099] Example 1: Preparation and identification of monoclonal antibodies

[0100] (I) Preparation of p72 protein

[0101] The present invention uses the B646L gene of the African swine fever virus HLJ / 18 strain (GenBank No. MK333180.1), and its coding sequence is as follows (SEQ ID NO.1):

[0102]

[0103] The p72 protein sequence encoded by the aforementioned B646L gene is as follows (SEQ ID NO.2):

[0104] MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPF GRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYY QPPLALWIKLRFWFNENVNLAIPSVSIPFGERFITIKLASQKDLVNEFPGLFVRQSRFIAGRPSRRNIRFKPWFIPGVINEISLTNNNELYINNLFVTPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISDQNPHQH RDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYST

[0105] The plasmid containing the aforementioned B646L gene coding sequence was transformed into Saccharomyces cerevisiae to induce the expression of foreign protein, and the obtained protein was purified by StrepⅡ purification system to obtain purified p72 protein. The operation was performed according to the method described in the following literature.

[0106] K.Meng,Y.Zhang,Q.Liu,Y.Huyan,W.Zhu,Y.Xiang,G.Meng, Structural Design and Assessing of Recombinantly Expressed African Swine Fever Virus p72 Trimerin Saccharomyces cerevisiae,Frontiers in Microbiology 13(2022).

[0107] (II) Preparation of monoclonal antibodies against p72 protein

[0108] The purified p72 protein solution (1 mg / ml) 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, with a total of 6 mice, and the immunization dose was 50 μg / mouse. Then the same dose of p72 protein solution was emulsified with Freund's incomplete adjuvant to prepare the immunogen, and two booster immunizations were performed, with an interval of 2 weeks between each immunization. Ten days after the second booster immunization, blood was collected from each mouse, serum was separated, and antibody titers were determined using the indirect p72-ELISA method. The mouse with the highest antibody titer was selected for the last booster immunization with purified p72 protein solution without adjuvant, with an immunization dose of 50 μg / mouse, euthanized after 3 days, and spleen cells were taken and fused with SP2 / 0 myeloma cells. The fused hybridoma cells were cultured in HAT selection culture for 10 days. Using the purified p72 protein prepared in step (a) as the coating antigen, the p72 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 capable of secreting antibodies against p72 protein was obtained. It was named cell line 2F11, and the monoclonal antibody secreted by the hybridoma cell was called 2F11.

[0109] (III) IFA identification of p72 protein monoclonal antibody

[0110] The reactivity of 2F11 with PAM and BK2258 cells infected with ASFV was detected by indirect immunofluorescence assay, respectively. The specific steps are as follows.

[0111] (1) Virus inoculation

[0112] ASFV SD / DY-I / 21 strain (GenBank No. MZ945537.1) was used as 10 3.80 TCID 50 / ml dose was inoculated into the well-grown PAM cell monolayer and BK2258 cell monolayer in 96-well plates, the culture medium was RPMI 1640 culture medium, the inoculation volume was 100 μl / well, and the culture was carried out at 37°C and 5% CO2 for 48 hours.

[0113] (2) Cell fixation

[0114] Discard the culture medium, wash twice with PBS, 250 μl / well, 1 min / time, and discard the PBS; add pre-cooled 4% (w / w) paraformaldehyde fixative (solvent is PBS), 100 μl / well, let stand at room temperature for 30 min, discard the fixative, wash twice with PBS, 250 μl / well, 1 min / time, and discard the PBS.

[0115] (3) Membrane treatment

[0116] Add 0.25% (v / v) Triton-X100 permeabilization solution (solvent is PBS), 200 μl / well, and let stand at room temperature for 15 min. Discard the permeabilization solution, and wash twice with PBS, 250 μl / well, 1 min / time, and discard PBS.

[0117] (4) Add primary antibody

[0118] For the two cell monolayers in the above 96-well plates, add one of the following three primary antibodies to different wells:

[0119] (i) 2F11 solution, concentration: 1 mg / ml, solvent: PBS, diluted with PBS at a volume ratio of 1:100.

[0120] (ii) Positive control: mouse polyclonal antibody against African swine fever virus p72 protein, diluted 1:200 in PBS.

[0121] (iii) Negative control: SP2 / 0 cell culture supernatant.

[0122] 100 μl / well, incubate at 37°C for 30 min. Discard the sample, wash with PBS 3 times, 250 μl / well, 1 min / time, and discard PBS.

[0123] (5) Add secondary antibody

[0124] Add FITC-labeled goat anti-mouse IgG, dilute with PBS at 1:100, add 100 μl / well after dilution, and incubate at 37°C for 30 min. Discard the secondary antibody, wash with PBS 3 times, 250 μl / well, 1 min / time, and do not discard the wash solution for the last wash.

[0125] (6) Result determination

[0126] Observe the 96-well plate with a fluorescence microscope and take photos. Select a field of view for each operation. Figure 1 As shown in Figure A, the test results of PAM cells and BK2258 cells are the same, with the monoclonal antibody 2F11 test wells showing green fluorescence, the negative control wells showing no green fluorescence, and the positive control wells showing green fluorescence. This shows that 2F11 can bind to the African swine fever virus that has infected the cells.

[0127] (IV) WB identification of p72 protein monoclonal antibody

[0128] The p72 protein purified in step (I) was used to detect the specificity of 2F11. The PAM cell lysate infected with ASFV SD / DY-I / 21 strain was used as a positive control, and the uninfected PAM cell lysate was used as a negative control. The primary antibody was the 2F11 solution in step (I), and the secondary antibody was IRDye 800CW-coupled goat anti-mouse IgG antibody. Conventional Western blot experiments were performed. Results are shown in Figure 1 B, where lane 1 is the PAM lysate infected with ASFV, and lane 2 is the PAM lysate that is not infected. It can be seen that the monoclonal antibody 2F11 has a strong reaction with the p72 protein and ASFV-infected cells, indicating that the prepared 2F11 is specific for the ASFV p72 protein.

[0129] (V) Typing of p72 protein monoclonal antibodies and gene sequence determination

[0130] 2F11 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.

[0131] The total RNA of hybridoma 2F11 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:

[0132] Heavy chain CDR1 coding sequence (SEQ ID NO.3):

[0133] AATACTAATTATTGCTGGCAC

[0134] Heavy chain CDR1 protein sequence (SEQ ID NO.4):

[0135] NTNYCWH

[0136] Heavy chain CDR2 coding sequence (SEQ ID NO.5:

[0137] CGCATATGTTATGAAGGTTCAGTATATTATAGTCCAGCCATCAAAAGC

[0138] Heavy chain CDR2 protein sequence (SEQ ID NO.6):

[0139] RICYEGSVYYSPAIKS

[0140] Heavy chain CDR3 coding sequence (SEQ ID NO.7):

[0141] GAAAACCATGGTTACTTCGATGTC

[0142] Heavy chain CDR3 protein sequence (SEQ ID NO.8):

[0143] ENHGYFD

[0144] Light chain CDR1 coding sequence (SEQ ID NO.9):

[0145] AGATCTAGTCAGAGCCTTATACACAGTCATGGAGACACCTATTTACAT

[0146] Light chain CDR1 protein sequence (SEQ ID NO.10):

[0147] RSSQSLIHSHGDTYLH

[0148] Light chain CDR2 coding sequence (SEQ ID NO.11):

[0149] AAAGTTTCCAACCGATTTTCT

[0150] Light chain CDR2 protein sequence (SEQ ID NO.12):

[0151] KVSNRFS

[0152] Light chain CDR3 coding sequence (SEQ ID NO.13):

[0153] TCTCAAAGTACACATGTTCCCACG

[0154] Light chain CDR3 protein sequence (SEQ ID NO.14):

[0155] SQSTHVPT

[0156] The hybridoma cell line 2F11 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: C202402; the culture name is African swine fever virus P72 monoclonal antibody hybridoma cell line 2F11 Hybridoma cellline 2F11; the Chinese classification name is: Hybridoma Cell; the English classification name is: Hybridoma Cell; the deposit time is January 5, 2024; the identified survival time is January 11, 2024.

[0157] Example 2: Evaluation of the potential application of p72 monoclonal antibody-bELISA and preliminary establishment of the method

[0158] In order to evaluate the application prospect of 2F11 as an ASFV antibody detection reagent, a bELISA method based on 2F11 was established. Eight 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 2F11 and establish the bELISA method.

[0159] The p72 protein bELISA antibody detection method was used to determine the p72 antibody blocking rate. The detection procedure is as follows:

[0160] 1. Coating: Dilute the yeast-expressed African swine fever virus p72 protein antigen prepared in Example 1 into 2 μg / ml antigen dilution with antigen coating solution (carbonate buffer, pH 8.0), add to 96-well plate, 100 μl / well, and coat overnight at 4°C.

[0161] 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.

[0162] 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.

[0163] 4. Washing: Same as 2.

[0164] 5. Sample addition: dilute each serum with PBS at a volume ratio of 1:10, add to a 96-well plate, 100 μl / well, and incubate at 37°C for 2 hours.

[0165] 6. Washing: Same as 2.

[0166] 7. Add antibody: dilute 2F11 (1 mg / ml) with PBS at a volume ratio of 1:1000, 100 μl / well, and incubate at 37°C for 2 hours.

[0167] 8. Washing: Same as 2.

[0168] 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, 100 μl / well, and incubate at 37°C for 45 minutes.

[0169] 10. Washing: Same as 2.

[0170] 11. Color development: Add 100 μl TMB substrate color development solution (Sigma, T0440-1L) to each well for color development and react at 37°C in the dark for 10 minutes.

[0171] 12. Stop: Add 50 μl stop solution (2M H2SO4) to each well.

[0172] 13. Reading: Measure OD within 5 minutes of stopping 450nm value.

[0173] According to the following formula, the blocking rate % = [(negative control average OD 450nm - Sample OD 450nm ) / mean OD of negative control 450nm ]×100.

[0174] bELISA blocking rate results refer to Figure 1 C, in the figure, the PI values ​​of ASFV-positive pig sera (n=8) and negative sera (n=4) are shown as points on two vertical axes (1 represents the negative sample result, 2 represents the positive sample result).

[0175] It can be seen that 2F11 can be blocked by ASFV-positive pig serum and shows a high inhibition rate. Next, the present invention further evaluated p72-2F11 and used it to establish a monoclonal antibody-based bELISA method for ASFV antibody detection.

[0176] Example 3: Optimization of p72-b ELISA reaction conditions

[0177] The optimal dilutions of HRP-labeled 2F11 and serum samples were determined by systematic checkerboard titration.

[0178] (I) Optimization of secondary antibody concentration

[0179] The affinity purified 2F11 was labeled with horseradish peroxidase to obtain 2F11-HRP (concentration 1 mg / ml, solvent PBS), and the 2F11-HRP was diluted with a secondary antibody diluent (PBS) at a volume ratio of 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:12000, 1:14000, 1:16000, 1:20000, 1:22000, and 1:24000. One of the 8 positive sera in Example 2 (diluted with PBS at a volume ratio of 1:10), one of the 4 negative sera in Example 2 (diluted with PBS at a volume ratio of 1:10), and the PBS solution were selected as the primary antibody in step 5. Step 7, step 8, and step 9 were combined into one step, specifically: for each of the aforementioned secondary antibody dilutions, 100 μl / well, incubation at 37° C. for 45 minutes, and other operating steps and parameters were the same as in Example 2 to perform a p72-b ELISA test.

[0180] Experimental results Figure 2 It can be seen from A that the OD of each test well of the positive serum is close to 0, and complete blocking has occurred. When the dilution of the 2F11-HRP antibody is 1:12000, the OD value of the negative serum and 2F11-HRP is still constant at around 2.0.

[0181] (II) Optimization of serum dilution

[0182] In order to optimize the dilution of pig serum, the present invention used PBS to dilute 5 known ASFV-positive and 4 known ASFV-negative pig sera at a total of 10 dilutions of 1:1, 1:2, 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640, respectively, and the fixed dilution (1:12000) of HRP-labeled 2F11 was selected. The other operating steps and parameters were the same as step (a) of Example 3, and the p72-b ELISA test was performed.

[0183] Blocking rate result record and Figure 2 B. It can be seen that a 1:1 serum dilution is optimal to establish a new bELISA.

[0184] (III) Optimization of incubation time

[0185] The serum sample and HRP monoclonal antibody 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 p72-b ELISA test was performed. The results showed that the effect of 30 min was the best.

[0186] Subsequent tests are all performed based on the conditions optimized in this step.

[0187] Example 4: Determination of p72-b ELISA criteria

[0188] 194 porcine serum samples were used to standardize the threshold value of p72-b ELISA positive and negative judgment, including 133 ASFV negative sera and 61 ASFV positive sera. The p72-b ELISA method was determined using the operating parameters optimized in Example 3.

[0189] The average PI value of the newly established b-ELISA for negative serum was calculated to be 8.4%. In order to ensure high specificity, the threshold was calculated by adding 3 times the standard deviation (6.5%) to the average PI value (8.4%) and set at 28%.

[0190] The statistical results of inhibition rate can be found in Figure 3 B, B, interactive dot-line graph of PI values ​​of pig serum samples when the threshold is 28%. In the figure, the PI values ​​of pig serum samples are represented by dots on 4 vertical axes, N is ASFV negative serum (133 samples); W is wild-type ASFVHLJ / 18 experimental infection serum (61 samples); F is field serum samples (164 samples); V is immune serum of pigs vaccinated with 7-gene deleted ASFV candidate attenuated vaccine (HLJ / 18-7GD) (the transformation process of HLJ / 18-7GD strain is recorded in the patent document of Chinese patent application No. 201910348878.7, in which the corresponding name is rASFVΔCD2V / 360-eGFP-mCherry strain) (19 samples).

[0191] According to the operating characteristic (ROC) analysis of the data, when the threshold of PI was 28%, the specificity of the test was 99.33% (95% confidence interval: 97.59% to 99.92%), and the sensitivity was 100% (95% confidence interval: 95.49% to 100.00%). The area under the curve (AUC) was 1.00, indicating that the test accuracy was high ( Figure 3 In A), ROC curve analysis shows the accuracy of the area under the curve (AUC = 1.00).

[0192] After standardization of the newly established p72-b ELISA, 164 field serum samples were tested. 162 samples were negative as expected, and only two samples had PI values ​​slightly higher than the critical value (31.7% and 31.4%) ( Figure 3 B). All 19 pig sera from clinical trials were ASFV serum positive, indicating that the bELISA method based on p72 monoclonal antibody has a high sensitivity for ASFV antibody detection.

[0193] Example 5: Evaluation of the ability of the established bELISA to detect antibodies infected with low-virulence genotype II variants and genotype I ASFV strains in China

[0194] Previous studies have shown that low-virulence genotype II variants and genotype I ASFV strains have appeared in China. Two natural low-virulence strains, ASFV genotype I attenuated strain SD / DY-I / 21 strain (GenBank: MZ945537) (n=6) and ASFV genotype II attenuated strain HLJ / HRB1 / 20 strain (GenBank: MW656282) (n=4), were used to infect experimental pigs. Serum was collected on days 0, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 after inoculation. The p72 b ELISA method optimized in Example 3 was used to detect the kinetics of serum antibody reactions to evaluate whether the established method has the potential to detect antibodies infected by these natural low-virulence strains.

[0195] Results Figure 3 C and D show that among the pigs infected with HLJ / HRB1 / 20, this method detected that the ASFV antibodies of two pigs turned positive from day 11, and the ASFV antibodies of another pig turned positive from day 13 ( Figure 3 On the other hand, ASFV antibodies detected by p72 bELISA turned positive as early as day 7 in one pig and day 9 in five pigs infected with SD / DY-I / 21 ( Figure 3 D). The p72 b ELISA method can detect the above two genotypes of ASFV antibodies and has certain application value in the epidemiological monitoring of African swine fever.

[0196] It can be seen that the p72-b ELISA method of the present invention can well detect the serum antibody status after infection with different strains such as SD / DY-I / 21 strain and HLJ / HRB1 / 20 strain, and can therefore be used in clinical practice.

[0197] Example 6: p72-b ELISA specificity test

[0198] The specificity of the p72-2F11-based bELISA was evaluated using positive pig sera against several other major pig viruses. Using the optimized conditions in Example 3, CSFV (classical swine fever virus) positive pig sera, PRRSV (porcine reproductive and respiratory syndrome virus) positive pig sera, PRV (pseudorabies virus) positive pig sera, FMDV / O (foot-and-mouth disease virus type O) positive pig sera, PEDV (porcine epidemic diarrhea virus) positive pig sera, TGEV (porcine transmissible gastroenteritis virus) positive pig sera, ASFV (African swine fever virus) positive pig sera were used as primary antibodies for detection, and the detection conditions were parallel. See the test results for details. Figure 4 ,It can be seen that the test results of other sera except ASFV-positive pig sera were all negative, and the PI value was significantly reduced.

[0199] The results showed that the p72-2F11-based bELISA had good specificity for the diagnosis of ASFV and had no cross-reaction with other porcine viruses.

[0200] The polyclonal antisera of 6 different swine pathogens were studied. The PI value of the anti-ASFV serum was the largest, at 97.6%, and the PI values ​​of the other anti-CSFV, PRRSV, PRV, FMDV / O, PEDV and TGEV sera were -0.7% to 8.1%. It can be seen that the p72-bELISA constructed by the present invention has good specificity for the detection of African swine fever virus antiserum and meets the requirements for clinical use.

[0201] Example 7: Repeatability test

[0202] Six ASFV positive sera and four ASFV negative sera were randomly selected, and three batches (enzyme-labeled plates coated with three purified proteins) of p72-b ELISA plates were used for detection, and the test was performed according to the operating parameters of Example 3. At the same time, the test was repeated three times in the same batch (three 96-well plates were selected in the same batch of coated plates) to evaluate, calculate the coefficient of variation of the blocking rate, and evaluate the inter-plate repeatability and intra-plate repeatability. The results showed that the coefficient of variation was less than 15% (Table 1), indicating that the established method is repeatable.

[0203] Table 1. p72-b ELISA repeatability test

[0204]

[0205] Example 8: Analysis of the coincidence rate between p72-bELISA and P72-iELISA

[0206] 507 serum samples (including local clinical serum samples and experimentally infected pig serum) were tested using both the p72-b ELISA method established by the present invention (operated according to the parameters of Example 3) and the commercialized p72-i ELISA.

[0207] The results showed that the sensitivity of the p72-b ELISA method of the present invention was 98.77%, the specificity was 97.97%, and the consistency rate with the p72-i ELISA method was 98.22% (Table 2). It can be seen that the p72-b ELISA method of the present invention has a good application prospect.

[0208] Table 2. Coincidence test between p72-bELISA and P72-iELISA

[0209] a: the number of samples with positive results in both P72-iELISA and p72-bELISA tests; b: the number of samples with positive results in p72-iELISA and negative results in p72-bELISA tests; c: the number of samples with negative results in P72-iELISA and positive results in p72-bELISA tests; d: the number of samples with negative results in both P72-iELISA and p72-bELISA tests; e: sensitivity % = a / (a+b)×100; f: specificity % = d / (c+d)×100; g: conformity % = (a+d) / (a+b+c+d)×100.

[0210] Example 9: First round identification of epitopes of p72-2F11

[0211] In order to map the antigenic epitope of p72-2F11, the protein sequence shown in SEQ ID NO. 2 in Example 1 is used as the benchmark for the full length of p72 protein. The present invention designed 6 truncated p72 recombinant fragments: p72-N (1-103 positions), p72-ER1 (104-195 positions), p72-ER 2 (199-332 positions), p72-ER 3 (354-419 positions), p72-ER 4 (465-543 positions) and p72-C (544-646 positions), which span the entire length of the p72 protein, see Figure 6 The upper part of A. Figure 6 Figure A shows the schematic diagram of the design of the full-length truncated gene fragment of the p72 protein and the identified epitopes.

[0212] Six recombinant p72 truncated proteins were expressed, of which p72-ER1 to p72-ER4 were cloned into pGEX-4T-1 vectors, and the short peptides were expressed as GST fusion proteins. p72-N and p72-C were cloned into pMAL-C5X vectors, and the short peptides were expressed as MBP fusion proteins. HRP-labeled anti-GST antibody and HRP-labeled anti-MBP antibody were used for detection, respectively. Western blot was used to analyze the expression of each fusion protein. The results are shown in Figure 2. Figure 5 As shown in A. Figure 5 As shown in Figure A, the six truncated p72 recombinant fragments can be expressed successfully, providing a basis for analyzing the binding characteristics of 2F11.

[0213] Furthermore, the linear epitopes were identified by immunoblotting (Western blot) of the above six fusion proteins using 2F11. The reactivity of the six p72 truncated fusion proteins (p72-N, p72-ER1, p72-ER2, p72-ER3, p72-ER4 and p72-C) with 2F11 is shown in Figure 6 C, P72-full length protein represents the p72 protein (wh) prepared in Example 1. The results showed that only the GST-tagged P72-ER2 protein reacted strongly with 2F11, indicating that the antigenic linear epitope of 2F11 is located in P72-ER2 (199-332aa).

[0214] Example 10: Second round of identification of the epitope of p72-2F11

[0215] According to the preliminary results of Example 9, p72-ER2 was further truncated into five partially overlapping short peptides (ER2-1 to ER2-5), each peptide differing from its adjacent peptide by 20 amino acids, with the start and end sites as shown in Figure 6 The coding sequences of the five short peptides were cloned into the pMAL-C5X vector, and the short peptides were expressed as MBP fusion proteins. The expression of each fusion protein was analyzed by Western blot in the same manner as in Example 9. The results are shown in FIG. Figure 5 As shown in B. Figure 5 As shown in Figure B, the five truncated p72 recombinant fragments can be expressed successfully, providing a basis for analyzing the binding characteristics of 2F11.

[0216] Furthermore, the above five fusion proteins were detected by Western blot using monoclonal antibody 2F11 in the same manner as in Example 9. The Western blot results are shown in Figure 6D, it can be seen that ER2-1 and ER2-2 react strongly with 2F11. Since ER2-1 and ER2-2 overlap by 20 amino acids, the epitope site of 2F11 is subtracted to the common sequence of the two fragments, i.e., 273 PKFLSQHFPENSHNIQTAGK 292 .

[0217] Example 11: Fine Mapping of 2F11 Target Epitope

[0218] In order to accurately locate the core sequence of the 2F11 epitope, the present invention deletes two amino acids at a time. 273 PKFLSQHFPENSHNIQTAGK 292 The start and end sites of the 17 peptides from P1 to P15 are shown in the table below. Figure 6 B. The coding sequences of 17 short peptides were cloned into pMAL-C5X vector, and the truncated peptides were expressed as MBP fusion products. The expression of each fusion protein was analyzed by Western blot in the same way as in Example 9. The results are shown in FIG. Figure 5 As shown in C. Figure 6 The peptide is shown in B 273 PKFLSQHFPENSHNIQTAGK 292 Sequential deletions of the N-terminal and C-terminal amino acids of (P1-P15) and their respective WB results for p72-2F11, with weak reactions indicated by asterisks (*). Figure 5 As can be seen from the figure, all 17 truncated p72 recombinant fragments can be successfully expressed, providing a basis for analyzing the binding characteristics of 2F11.

[0219] Furthermore, the 17 fusion proteins were detected by Western blot using 2F11 in the same manner as in Example 9. The Western blot results are shown in Figure 6 The results showed that the deletion of the four amino acid residues at the C-terminus did not affect the reactivity of the monoclonal antibody 2F11. However, when the other two amino acids (I 287 Q 288 ), the reactivity of 2F11 to polypeptide-3 (P3) was significantly reduced. On the other hand, after the continuous removal of 10 amino acids at the N-terminus, the peptide fusion protein still maintained reactivity with 2F11. However, when the asparagine at position 283 (N 283 ) was removed from the N-terminus, the resulting fusion protein (P13) completely lost its reactivity to 2F11. These results indicate that 283 NSHNIQ 288 It is the core sequence recognized by 2F11 (SEQ ID NO.15).

[0220] Example 12: Homology analysis of identified p72 epitopes

[0221] To evaluate whether the epitope region is conserved between different genotypes of ASFV, the present invention analyzed the p72 protein sequences of 23 ASFV isolates, including 11 genotypes. Sequence alignment of these 23 ASFV isolates showed that the epitope identified by the monoclonal antibody 2F11 obtained in this study ( 283 NSHNIQ 288 )Highly conserved (e.g. Figure 7 A).

[0222] Figure 7 Figure 4 shows the linear B cell epitope characterization analysis of p72 protein, and A shows the identified antigen epitope ( 283 NSHNIQ 288 ) was compared with 23 ASFV reference strains for homology, and the identified antigen epitope sequences are indicated by black boxes. B is the three-dimensional structure of p72 protein simulated by PyMOL software. 283 NSHNIQ 288 Epitopes recognized in the monomeric p72 structure (left) and the epitopes recognized in the trimeric structure (right) are indicated by horizontal lines.

[0223] It can be seen that epitopes ( 283 NSHNIQ 288 ) in GenBank with the numbers QBH90570.1, AXP99042.1, QIE06872.1, AYW34053.1, QDL88113.1, QIA61496.1, QLF78610.1, QST88166.1, CAD2068444.1, QGJ83444.1, QTP96390.1, UEN73249.1, UEN73091.1, and CAN10 431.1, WEG42281.1, AJL34260.1, NC_044945.1, NC_044952 p72 proteins and GenBank No. AY261365.1, AY261366.1, AY261364.1, NC_044953.1, NC_044954.1 genome obtained by sequence analysis are the same, and the sequence is highly conserved in different virus strains. It can be seen that the 2F11 obtained by the present invention has efficient recognition specificity for at least these p72 proteins and has a large spectrum of African swine fever virus strains. Epitope ( 283 NSHNIQ 288) are located on the outer surface of the protein structure of the monomer and trimer, which are easy to connect with immune cells and stimulate humoral immune response. It also shows that 2F11 can recognize the surface domain of p72 protein and has the ability to efficiently recognize p72 protein.

[0224] summary

[0225] P72 is the main capsid protein of ASFV, one of the most antigenic and immunogenic proteins, and is highly conserved between different ASFV isolates. These unique properties make p72 one of the most important antigenic targets to advance diagnostic tests and vaccine development. bELISA is a specific antibody detection method that has been widely used to diagnose human diseases and monitor animal infections. The present invention establishes a hybridoma cell line 2F11 that stably secretes anti-p72 monoclonal antibodies, and detects the ability of the secreted antibody 2F11 to compete with ASFV positive serum for binding to the p72 antigen. Finally, a method for detecting African swine fever antibodies using bELISA based on p72 monoclonal antibodies was established. 2F11 is coupled to horseradish peroxidase (HRP) to detect antibodies, eliminating the step of coupling anti-mouse secondary antibodies with horseradish peroxidase (HRP), shortening the detection time and reducing costs. Compared with commercially available indirect ELISA, the bELISA established by the present invention has a higher compliance rate and a fast detection speed. p72-bELISA and p72 indirect ELISA (p72-iELISA) were used in parallel to test 164 clinical samples, and the results showed good consistency.

[0226] With the emergence of low-virulence strains of ASFV (genotypes I and II) in China, antibody detection methods have received increasing attention. Therefore, diagnostic methods that can accurately detect antibodies to low-virulence genotypes I and II ASFV are urgently needed. To further validate the p72-bELISA method, low-virulence genotypes I and II ASFV positive sera were tested, and the sensitivity of this method for detecting ASFV antibodies was the same as that of the commercially available p72-iELISA kit.

[0227] Identification of B cell epitopes in viral structural proteins is crucial for understanding virus-host interactions and developing effective diagnostic tools and vaccines. Due to the complex structure of the p72 trimer, information about p72-specific epitopes is still limited and is still being explored. In order to identify the linear epitopes recognized by 2F11, a series of truncated overlapping peptides covering the full length of the B646L / p72 gene were expressed as MBP or GST fusion proteins for peptide scanning. Notably, 2F11 recognized a short truncated p72 fusion protein located between amino acids 199 and 332 of the p72 protein. Based on the strategy of expressing truncated peptides as fusion proteins, the shortest core sequence that can be recognized by monoclonal antibody 2F11 is 283NSHNIQ 288 Sequence alignment analysis showed that the epitope sequence was highly conserved among different ASFV strains.

[0228] In summary, the present invention prepared a monoclonal antibody 2F11 against p72 trimer protein and established bELISA based on it. This method has good sensitivity, specificity and repeatability and can be used for serological monitoring of ASFV infection. A new linear B cell epitope of -p72 was further identified. 283 NSHNIQ 288 This study is not only of great significance for the serological diagnosis of ASFV, but also lays the foundation for further research on the antigenic function of p72 protein.

[0229] 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 microbial preservation number of the hybridoma cell is CCTCC NO: C202402.

2. A monoclonal antibody, which is a monoclonal antibody secreted by the hybridoma cell of claim 1 or a monoclonal antibody secreted by a subculture cell of the hybridoma cell of claim 1.

3. A monoclonal antibody, wherein the monoclonal antibody retains specific binding activity to African swine fever virus p72 protein, and the monoclonal antibody comprises a heavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, 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 EQ ID NO.

14.

4. The monoclonal antibody according to claim 3, characterized in that The monoclonal antibody is a porcine monoclonal antibody.

5. The monoclonal antibody according to claim 3, characterized in that The amino acid sequence of the African swine fever p72 protein is shown in SEQ ID NO.

2.

6. An antibody derivative, which retains specific binding activity to African swine fever virus p72 protein, and the antibody portion of the antibody derivative is a monoclonal antibody as described in any one of claims 3-5; the form of the antibody derivative is selected from: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, porcine antibodies, single-chain antibodies, chimeric monoclonal antibodies and modified monoclonal antibodies.

7. The antibody derivative according to claim 6, characterized in that The amino acid sequence of the African swine fever p72 protein is shown in SEQ ID NO.

2.

8. A fusion protein, which retains specific binding activity to African swine fever virus p72 protein, and comprises the monoclonal antibody according to any one of claims 3 to 5 and a tag peptide for separating and purifying the protein.

9. The fusion protein according to claim 8, characterized in that The amino acid sequence of the African swine fever p72 protein is shown in SEQ ID NO.

2.

10. A gene expression cassette, wherein the coding sequence in the gene expression cassette can encode the monoclonal antibody according to claim 2, the monoclonal antibody according to any one of claims 3 to 5, or the fusion protein according to claim 8 or 9.

11. A genetic engineering expression vector, comprising the gene expression cassette according to claim 10.

12. A host cell, comprising the genetically engineered expression vector according to claim 11, wherein the encoded protein in the expression cassette of the genetically engineered expression vector is constitutively expressed or artificially induced.

13. An ELISA kit, comprising a microplate, wherein the bottom surface of the wells of the microplate is coated with a polypeptide or a fusion protein; The amino acid sequence of the polypeptide is shown in SEQ ID NO.15; The amino acid sequence of the fusion protein consists of a polypeptide with an amino acid sequence as shown in SEQ ID NO.15 and a tag peptide for separating and purifying the protein; The kit also contains the monoclonal antibody of claim 2 labeled with horseradish peroxidase, the monoclonal antibody of any one of claims 3-5 labeled with horseradish peroxidase, the antibody derivative of claim 6 or 7 labeled with horseradish peroxidase, or the fusion protein of claim 8 or 9 labeled with horseradish peroxidase.

14. The ELISA kit according to claim 13, wherein In the step of preparing the microplate, the concentration of the polypeptide or the fusion protein in the coating solution is 1-3 μg / ml, and the coating amount per well is 50-200 μl; and / or The kit also contains African swine fever virus-negative pig serum and / or African swine fever virus-positive pig serum.

15. A kit, comprising the hybridoma cell according to claim 1, the monoclonal antibody according to claim 2, the monoclonal antibody according to any one of claims 3 to 5, the antibody derivative according to claim 6 or 7, the fusion protein according to claim 8 or 9, the genetically engineered expression vector according to claim 11, or the host cell according to claim 12.

16. A method for identifying African swine fever virus for non-diagnostic purposes, the method comprising the following steps: S1: Infect African swine fever virus susceptible cells with the sample to be tested to obtain infected cells; S2: fixing the infected cells on a solid surface to obtain immobilized cells; S3: adding the monoclonal antibody according to claim 2 or the monoclonal antibody according to any one of claims 3 to 5 to the immobilized cells for incubation to obtain immobilized cells for the first incubation; S4: adding the monoclonal antibody specific binding substance connected with the label to the fixed cells of the first incubation for incubation to obtain the fixed cells of the second incubation; S5: Characterizing the marker indirectly bound to the fixed cells in the second incubation, and determining whether African swine fever virus exists in the sample to be tested based on the presence or absence of the marker.

17. The method according to claim 16, characterized in that In step S1, the African swine fever virus susceptible cells are wild boar kidney cells or primary porcine alveolar macrophages with a microbial collection number of CCTCC NO: C2022258; and / or In step S2, the solid surface is the inner surface of a well of a microplate; and / or In step S4, the monoclonal antibody specific binding substance connected with a label is a secondary antibody against the monoclonal antibody according to claim 2 or the monoclonal antibody according to any one of claims 3 to 5 labeled with a fluorescent molecule; and / or In step S5, the fixed cells incubated for the second time are photographed using a fluorescence microscope, and it is determined whether African swine fever virus exists in the sample to be tested based on the photographic results.

18. A method for identifying antibodies to African swine fever virus for non-diagnostic purposes, the method comprising the following steps: P1: coating a solid surface with African swine fever p72 protein, an African swine fever p72 protein fusion protein containing a tag peptide for separating and purifying the protein, a polypeptide or a fusion protein to obtain a coated solid surface; The amino acid sequence of the polypeptide is shown in SEQ ID NO.15; The amino acid sequence of the fusion protein consists of a polypeptide with an amino acid sequence as shown in SEQ ID NO.15 and a tag peptide for separating and purifying the protein; 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 of claim 2 connected with a label, the monoclonal antibody of any one of claims 3 to 5 connected with a label, or the antibody derivative of claim 6 or 7 connected with a label to the solid phase surface of the first incubation for incubation to obtain a solid phase surface of the second incubation; P4: Characterize the label that is indirectly bound to the solid surface of the second incubation, and determine whether African swine fever virus antibodies are present in the sample to be tested based on the presence or absence of the label.

19. The method according to claim 18, characterized in that In step P1, the amino acid sequence of the African swine fever p72 protein is as shown in SEQ ID NO.2; and / or In step P1, the solid surface is the inner surface of a well of a microplate; and / or In step P2, African swine fever virus-negative pig serum is added in parallel as a negative control; and / or African swine fever virus-positive pig serum is added in parallel as a positive control; and / or In step P3, the marker is horseradish peroxidase. In step P4, TMB substrate colorimetric solution is added to the solid surface of the second incubation. The OD value of the solid surface of the second incubation is measured. 450nm The value is calculated to determine whether African swine fever virus antibodies exist in the sample to be tested.

20. The method of claim 18, wherein: In step P2, the African swine fever virus-negative pig serum is diluted with a PBS solution at a volume ratio of 1:1 to 1:10 before use, and / or the African swine fever virus-positive pig serum is diluted with a PBS solution at a volume ratio of 1:1 to 1:10 before use.

21. The method of claim 18, wherein: In step P3, the label is horseradish peroxidase, and the solvent of the monoclonal antibody of claim 2 connected with the label, the monoclonal antibody of any one of claims 3-5 connected with the label, or the antibody derivative of claim 6 or 7 connected with the label is PBS solution, and the concentration is 50-200 μg / L; and / or The identification is performed using the ELISA kit according to claim 13 or 14.

22. Use of the hybridoma cell according to claim 1, the monoclonal antibody according to claim 2, the monoclonal antibody according to any one of claims 3-5, the antibody derivative according to claim 6 or 7, the fusion protein according to claim 8 or 9, the gene expression cassette according to claim 10, the genetically engineered expression vector according to claim 11 or the host cell according to claim 12 in the preparation of a preparation for detecting African swine fever antibodies or African swine fever antigens.

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