Monoclonal antibody against African swine fever virus p72 protein and its application

Abstract

The present invention discloses an anti-African swine fever virus p72 protein monoclonal antibody and its application, belonging to the technical field of monoclonal antibodies. The present invention provides an anti-African swine fever virus monoclonal antibody 28H1, wherein the heavy chain variable region of the monoclonal antibody 28H1 includes a CDR1 with an amino acid sequence of EYPIH, a CDR2 with an amino acid sequence of IISTKYNNAGYNQKIKG, and a CDR3 with an amino acid sequence of NGGYLGDY; the light chain variable region of the monoclonal antibody 28H1 includes a CDR1 with an amino acid sequence of KADQNMGINVA, a CDR2 with an amino acid sequence of SAPYRDS, and a CDR3 with an amino acid sequence of QRYNAFPYT. The antibody of the present invention has the characteristics of high purity, strong affinity, good specificity, etc., and can be widely used in immunoassay technology.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to anti-African swine fever virus p72 protein monoclonal antibodies and applications thereof. Background Art

[0002] African swine fever (ASF) caused by African swine fever virus (ASFV) infection is an acute, febrile and highly contagious disease. ASFV is the only member of the Asfrican virus family and the only known arbovirus. The virus particle consists of five parts from the inside to the outside: the viral nucleoid genome, the inner shell, the inner membrane, the capsid and the outer membrane. The genome is a linear double-stranded DNA of 170-190 kb, encoding more than 151-167 proteins, including a variety of structural and non-structural proteins.

[0003] ASFV can be transmitted between wild boars and soft ticks through feeding, and rarely directly between wild boars. Once introduced into domestic pig populations, ASFV can be transmitted directly between pigs and cause a mortality rate close to 42%. Typical clinical signs include high fever, hemorrhagic lesions, cyanosis, anorexia, and ataxia. Different organs show severe vascular changes in acute and subacute forms of ASF, such as renal petechiae and diffuse hemorrhages in lymph nodes, pulmonary edema, disseminated intravascular coagulation, and thrombocytopenia. In order to control the virus, all pigs in the area are slaughtered, resulting in huge economic losses. However, there is currently no commercial vaccine or drug that can effectively control the spread and prevalence of the disease.

[0004] The p72 protein is the main component of the viral icosahedral capsid, encoded by the B646L gene, and accounts for about one-third of the total mass of the virus particle. The large proportion of p72 in the virus particle makes it one of the main antigens detected in infected pigs. In addition, p72 is a highly immunogenic protein. Therefore, it has been used as a target for the diagnosis of ASFV infection. Although there are technologies that can express p72 proteins through prokaryotic and eukaryotic systems, their natural structures and modifications are very different from the natural structures of proteins in virus particles, resulting in antibodies screened using prokaryotic proteins that do not react well with virus particles.

[0005] At present, there is no effective vaccine or drug available for ASFV. Detection technology plays an important role in ASFV prevention and control. The use of antibodies that are more in line with the natural structure of viral proteins can greatly improve the sensitivity of the detection method. The present invention prepares monoclonal antibodies by immunizing mice with inactivated African swine fever virus. A hybridoma cell line secreting p72 monoclonal antibodies was obtained by screening and subcloning purification. The monoclonal antibody secreted by the hybridoma cell line can be used to identify p72 protein in Western-Blot, and can also specifically identify p72 protein in ASFV infected cells in indirect immunofluorescence experiments. Provide a solid foundation for the study of the structure and function of ASFV p72 protein and the antigen-antibody detection method based on p72 protein. Summary of the invention

[0006] The purpose of the present invention is to provide monoclonal antibodies against African swine fever virus p72 protein and their applications to solve the problems existing in the above-mentioned prior art. The monoclonal antibodies against ASFV p72 protein prepared by the present invention have the advantages of high antibody purity, strong affinity and good specificity, and provide a basis for the study of the structure and function of ASFV p72 protein and the detection method based on p72 protein.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an anti-African swine fever virus p72 protein monoclonal antibody 28H1, wherein the heavy chain variable region of the monoclonal antibody 28H1 comprises a CDR1 with an amino acid sequence of EYPIH, a CDR2 with an amino acid sequence of IISTKYNNAGYNQKIKG, and a CDR3 with an amino acid sequence of NGGYLGDY; the light chain variable region of the monoclonal antibody 28H1 comprises a CDR1 with an amino acid sequence of KADQNMGINVA, a CDR2 with an amino acid sequence of SAPYRDS, and a CDR3 with an amino acid sequence of QRYNAFPYT.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 28H1 is shown in SEQ ID NO: 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody 28H1 is shown in SEQ ID NO: 2.

[0010] Furthermore, the monoclonal antibody 28H1 produces a specific immune response with African swine fever virus or its p72 protein antigen.

[0011] Furthermore, the heavy chain subtype of the monoclonal antibody 28H1 is IgG1, and the light chain subtype is Kappa.

[0012] The present invention also provides a nucleic acid molecule, which is a nucleic acid molecule encoding the monoclonal antibody 28H1.

[0013] The present invention also provides a vector, which comprises the nucleic acid molecule.

[0014] The present invention also provides a host cell, which comprises the nucleic acid molecule or the vector.

[0015] The present invention also provides the use of the monoclonal antibody 28H1 or the nucleic acid molecule or the vector or the host cell in preparing a detection product for African swine fever virus or its p72 protein.

[0016] Furthermore, the detection product comprises a reagent or a kit for detection using Western-Blot or indirect immunofluorescence method.

[0017] The present invention also provides a product for detecting African swine fever virus or its p72 protein antigen, wherein the product comprises the monoclonal antibody 28H1 or the nucleic acid molecule or the vector or the host cell.

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

[0019] The immunogenic protein used in the present invention is inactivated African swine fever virus, which makes the immune protein closer to the natural structure of the viral protein and gives the antibody the advantages of strong affinity and specificity.

[0020] The present invention provides a monoclonal antibody that can react specifically with ASFV and ASFV p72 protein, has good immunogenicity, good specificity, high sensitivity, can be widely used in immunoassay technology, and can be used as an antibody for Western-Blot and indirect immunofluorescence test to identify ASFV and p72 protein and determine the ASFV virus content. The present invention lays an important foundation for the clinical detection of ASFV and its p72 protein antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is the ELISA result diagram of the reaction between the inactivated ASFV virus and the African swine fever virus-positive pig serum in clinical practice in Example 1; wherein the P30 protein antigen is a positive control;

[0023] Figure 2The ELISA test results of serum antibody titer of immunized mice in Example 1;

[0024] Figure 3 This is the result of IFA analysis of ASFVp72 monoclonal antibody in Example 2 (p72 protein expressing cells);

[0025] Figure 4 The figure is the result of Western-Blot analysis of ASFV p72 monoclonal antibody in Example 2, pcaggs-p72: p72 eukaryotic expression protein, pcaggs: negative control;

[0026] Figure 5 This is the result of IFA analysis of ASFVp72 monoclonal antibody in Example 2 (virus-infected cells);

[0027] Figure 6 The figure is the result of Western-Blot analysis of ASFV p72 monoclonal antibody in Example 2, virus: cells infected with African swine fever virus, mock: blank cell control;

[0028] Figure 7 This is a diagram showing the SDS-PAGE detection result of the ASFVp72 monoclonal antibody purification effect in Example 3;

[0029] Figure 8 It is the titer diagram of ASFVp72 monoclonal antibody; NC is the negative control;

[0030] Fig. 9 This is the Western-Blot result of ASFV p72 monoclonal antibody fishing for p72 protein in Example 3, anti-p72: p72 antibody fishing, mIgG: mouse IgG fishing;

[0031] Fig.10 The results of PCR amplification of the variable region in Example 4, M: Marker, VL: light chain of monoclonal antibody 28H1, VH: heavy chain of monoclonal antibody 28H1. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1

[0038] 1. Preparation of immunogen

[0039] 1.1 ASFV inactivated virus particle concentration

[0040] The formaldehyde-inactivated virus solution was added to the ultracentrifuge tube, and a 30% (mass-to-volume ratio) sucrose solution was added to the bottom of the ultracentrifuge tube (roughly to the arc position) using a long syringe. After balancing, the virus was concentrated using an ultrahigh-speed refrigerated centrifuge (Beckman Optima XE-90), dissolved with PBS, and the protein content was determined using NanoDrop.

[0041] 1.2 Verification of the reactivity of concentrated virus particles - indirect ELISA test

[0042] 10 μg / well of the concentrated inactivated virus in 1.1 and 1 μg / well of the P30 protein stored in the laboratory were coated on the ELISA plate at 4°C overnight, 5% skimmed milk powder was used to block the ELISA plate at room temperature for 4 h, and the ELISA plate was washed with PBST buffer. The immunized mouse serum was diluted from 1:40 and used as the primary antibody for incubation at room temperature for 2 h. The ELISA plate was washed with PBST buffer to wash away the unbound primary antibody, and HRP-labeled goat anti-mouse was diluted 1:6000 as the secondary antibody for incubation at room temperature for 45 min. The ELISA plate was washed with PBST buffer, 100 μL / well of the color developing solution was added and incubated at 37°C incubator for 10 min, and then 50 μL / well of the stop solution was added, and the results were observed by a UV spectrophotometer at 450 nm.

[0043] The results of ELISA experiments showed that (see Figure 1 ), the inactivated virus has good reactogenicity and can be used as an immunogen to immunize mice.

[0044] Preparation of monoclonal antibody against 2ASFVp72 protein

[0045] 2.1 Immunization of mice

[0046] Five BALB / c female mice were taken and the inactivated virus protein was used to immunize the mice at a dose of 100 μg / mouse. For the first immunization, the inactivated virus and Freund's complete adjuvant were emulsified at an equal volume of 1:1 and the mice were immunized by subcutaneous injection at multiple points on the back. Two weeks later, Freund's incomplete adjuvant and inactivated virus were emulsified at an equal volume of 1:1 and immunized once, and then immunized once again after an interval of 2 weeks, for a total of 3 immunizations. 15 days after the third immunization, the tail vein blood of the immunized mice was collected.

[0047] 2.2 Indirect ELISA to detect serum titers of immunized mice

[0048] The ELISA plate was coated with 10 μg / well of inactivated viral protein at 4°C overnight, and the ELISA plate was blocked with 5% skim milk powder at room temperature for 4 hours. The ELISA plate was washed with PBST buffer, and the immunized mouse serum was diluted from 1:100 to 1:12800 as the primary antibody and incubated at room temperature for 2 hours. The ELISA plate was washed with PBST buffer to wash away the unbound primary antibody, and HRP-labeled goat anti-mouse was diluted 1:6000 as the secondary antibody and incubated at room temperature for 45 minutes. The ELISA plate was washed with PBST buffer, 100 μL / well of color development solution was added and incubated in a 37°C incubator for 10 minutes, and then 50 μL / well of stop solution was added, and the results were observed by a 450nm UV spectrophotometer.

[0049] The results are as follows Figure 2As shown, after mice were immunized with inactivated ASFV virus, antibodies against African swine fever virus proteins were detected in the mouse serum 15 days after the third immunization, and the subsequent cell fusion test could be carried out.

[0050] 2.3 Cell fusion

[0051] The spleen cells of mice after booster immunization were taken and fused with sp2 / 0 cells at a ratio of (10-15):1 in a 37°C water bath. PEG4000 was added for 1 min, and the cells were allowed to stand for 1 min. Then, 1 mL of 1640 medium was added for 1 min, and 1 mL of 1640 medium was added for 20 s. Then, 30 mL of medium was added for 3 min. The cells were centrifuged at 800 rpm for 5 min, and 1×HAT 20% FBS 1640 medium was used for plating. When the confluence of the successfully fused cells reached more than 50%, positive cell lines were screened.

[0052] 2.4 Hybridoma cell screening and cloning

[0053] LLC-PK1 cells inoculated with ASFV virus solution were used for indirect immunofluorescence test, and the cell culture supernatant 10 days after fusion was collected for indirect immunofluorescence test to screen positive cell clones. Finally, hybridoma cell wells that reacted positively with ASFV infected cells and negatively with LLC-PK1 blank cells were selected, and subcloned by limiting dilution method. After two subclones and indirect immunofluorescence test screening, a hybridoma cell line 28H1 that stably secreted monoclonal antibodies against African swine fever virus protein was obtained.

[0054] Example 2 Verification of the characteristics of hybridoma cells

[0055] 1 Identification of the reactivity of hybridoma supernatant with ASFV-p72 protein

[0056] 1.1 Construction of eukaryotic expression plasmid for African swine fever p72 protein

[0057] Using the African swine fever genome as a template, specific primers (see Table 1) were used to amplify the p72 gene with a length of 1935 bp. After double digestion with BamHI and NheI, it was connected to the pcaggs eukaryotic expression vector. The plasmid construction was verified successfully through plasmid sequencing and specific primer amplification identification.

[0058] Table 1 Primers for p72 amplification

[0059] Primer name sequence p72-F CTAGCTAGCGCCAGCGGGGGCGCCTTCTGCC p72-R CGCGGATCCTGTGCTGTACCTTAGCACGGCGC

[0060] 1.2 Indirect fluorescent immunoassay

[0061] HEK-293T cells were grown to a confluency of 80-90%, and then transfected with p72 protein eukaryotic vector expression plasmid. After 28-30 hours, indirect immunofluorescence test was performed. After discarding the cell culture medium, the cells were washed once with PBS, and then fixed with 4% paraformaldehyde at room temperature for 15 minutes. The fixative was discarded, and the cells were washed with PBS three times. PBS containing 0.1% Triton X-100 was used as permeabilization solution. After permeabilization at room temperature for 15 minutes, the permeabilization solution was discarded. After washing with PBS three times, PBS containing 5% BSA was used as blocking solution. The cells were blocked at room temperature for 2 hours, and the blocking solution was discarded. The hybridoma cell culture supernatant and HA antibody were added as primary antibodies. After incubation at room temperature for 2 hours, the cells were washed with PBST three times, each for 5 minutes. AlexaFlour 488 donkey anti-mouse IgG (H+L) was added at a dilution of 1:500. After incubation at room temperature for 45 minutes, the cells were washed with PBST three times, each for 5 minutes. After adding ready-to-use DAPI to stain the cell nucleus for 5 minutes, the DAPI was discarded, and the cells were washed with PBS three times. The results were observed under a fluorescence microscope.

[0062] Observation under an inverted fluorescence microscope showed that the virus reacted with the positive serum and the positive hybridoma supernatant in a specific antigen-antibody reaction. After adding the fluorescent secondary antibody, obvious specific fluorescence appeared in the field of view, and there was no fluorescence in the blank control well. The results are as follows: Figure 3 As shown, this indicates that the antibody can specifically react with the p72 protein of ASFV.

[0063] 1.3 Western-Blot

[0064] Collect HEK 293T cell samples 28 hours after pcaggs-p72 transfection, pre-treat and separate by SDS-PAGE, take a piece of film containing the target band and place it on the membrane transfer instrument, and transfer the membrane at 200mA for 1.5h. Block the PVDF membrane with 10% skim milk at room temperature for 2h. Wash 3 times with PBST for 5min each time, add hybridoma supernatant as primary antibody, and react at 4℃ with slow shaking overnight. Wash 3 times with PBST for 5min each time, incubate with HRP-labeled rabbit anti-mouse IgG secondary antibody (1:5000) at room temperature for 45min, and finally develop with ECL to observe the results. See the results. Figure 4 It can be seen that the ASFV p72 monoclonal antibody can effectively bind to the p72 eukaryotic expression protein, indicating that the antibody can react with the linearized p72 protein.

[0065] 2. Identification of fusion hybridoma cells and ASFV reactivity

[0066] 2.1 Indirect fluorescence immunoassay

[0067] LLC-PK1 cells were digested with trypsin and plated in 24-well cell plates. When the cells grew to about 90% confluency, ASFV virus solution was inoculated and incubated at 37°C for 2 hours. Fresh 2% DMEM was then used to continue culturing. After 36 hours of culturing at 37°C, indirect immunofluorescence assay was performed. After discarding the cell culture medium, wash twice with PBS, add 4% paraformaldehyde to fix at room temperature for 15 minutes, discard the fixative, wash 3 times with PBS, use PBS containing 0.1% Triton 100 as permeabilization solution, permeabilize at room temperature for 15 minutes, discard the permeabilization solution, wash 3 times with PBS, switch to PBS containing 5% BSA as blocking solution, block at room temperature for 2 hours, discard the blocking solution, add hybridoma supernatant as primary antibody, incubate at room temperature for 2 hours, wash 3 times with PBST, 5 minutes each time, add Alexa Flour488 donkey anti-mouse IgG (H+L) (1:500 dilution), incubate at room temperature for 45 minutes, wash 3 times with PBST, 5 minutes each time, add ready-to-use DAPI to stain the cell nucleus for 5 minutes, discard DAPI, add PBS to wash 3 times, and observe the results under a fluorescence microscope. Inverted fluorescence microscope observation shows that the virus ASFV reacts specifically with the monoclonal antibody. After adding the fluorescent secondary antibody, obvious specific fluorescence appears under the field of view. The results are as follows Figure 5 shown.

[0068] 2.2 Western-Blot

[0069] Collect LLC-PK1 cell samples 36 hours after inoculation with ASFV virus liquid, pre-treat and separate them by SDS-PAGE, take a piece of film containing the target band and place it on the membrane transfer instrument, and transfer the membrane at 200mA for 1.5 hours. Block the PVDF membrane with 10% skim milk at room temperature for 2 hours. Wash 3 times with PBST for 5 minutes each time, add hybridoma supernatant as primary antibody, and react at 4°C with slow shaking overnight. Wash 3 times with PBST for 5 minutes each time, incubate with HRP-labeled rabbit anti-mouse IgG secondary antibody (1:5000) at room temperature for 45 minutes, and finally develop with ECL to observe the results. Western-Blot identification results are as follows Figure 6 As shown. The results showed that the target band appeared at about 72 kDa in the lane, indicating that the prepared monoclonal antibody can produce a specific immune response with the ASFV virus.

[0070] 3 Identification of monoclonal antibody types and subclasses

[0071] The subtype of p72 monoclonal antibody was identified using Mouse Mecrodinal Antibody Isotyping Elisa Kit (purchased from Proteintech). Hybridoma cells identified as positive by indirect ELISA were subcloned, and after two rounds of subcloning, the supernatant of the subcloned cells was collected.

[0072] The antibody type was detected using a kit, and the results showed that a monoclonal antibody cell line that stably secreted ASFV p72 protein was successfully obtained, and the cell line secreted IgG1 type antibodies (Table 2), named 28H1.

[0073] Table 2 Identification of monoclonal antibody types and subclasses

[0074] Monoclonal antibodies IgG1 IgG2a IgG2b IgG2c IgG3 IgM Kappa Lambda 28H1 + - - - - - + -

[0075] Note: + indicates a positive reaction, - indicates a negative reaction.

[0076] As shown in Table 2, the heavy chain subtype of monoclonal antibody 28H1 is IgG1, and the light chain subtype is Kappa.

[0077] Example 3 Preparation of monoclonal antibodies by hybridoma cells

[0078] 1 Preparation and purification of ascites

[0079] Three female BALB / c mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Ten days later, approximately 2 × 10 6 Monoclonal cells in the logarithmic proliferation phase were injected into the peritoneal cavity of mice. After the abdomen of the mouse was significantly swollen, the ascites was extracted and the supernatant was collected by centrifugation at 10000r / min for 10min. The monoclonal antibody was purified using ProteinA / G affinity chromatography.

[0080] The results are as follows Figure 7 As shown in the figure, the antibody protein secreted by the monoclonal antibody cell line purified by ProteinA / G affinity chromatography showed two bands, one for the antibody heavy chain (55kDa) and the other for the antibody light chain (25kDa), and there was no other impurity protein in the background, indicating a good purification effect.

[0081] 2 Ascites titer detection

[0082] The monoclonal antibody 28H1 purified from ascites and irrelevant antibody (NC group) were serially diluted with PBST at 2 ng / μL, with a total of 8 dilution gradients. 50 μL / well of the diluted antibody was added to the ELISA plate coated with p72 polypeptide and acted at RT for 2 hours. After washing four times with PBST, HRP-labeled goat anti-mouse IgG (1:6000 dilution) was added and acted at RT for 45 minutes. After washing four times with PBST, 100 μL / well of colorimetric solution was added and incubated in a 37°C incubator for 10 minutes, and then 50 μL / well of stop solution was added, and the results were observed by a UV spectrophotometer at 450 nm.

[0083] The results are as follows Figure 8 As shown, when the monoclonal antibody 28H1 was diluted to 0.015625 ng / μL, its OD450 It was still greater than that of the NC group, indicating that the antibody titer could reach above 0.015625 ng / μL.

[0084] 3 Antibody-based IP assay

[0085] PAMs were inoculated in a 6-well plate and cultured in an incubator for 12 hours before inoculation with African swine fever virus. 24 hours after virus infection, RIPA-lysed cell samples were collected, the cell lysate supernatant was taken, p72 antibody and mouse IgG (negative control) coupled with A+G agarose were added, and the agarose enriched samples were collected for Western-Blot detection after incubation at 4°C for 12 hours. The Western-Blot results are shown in Figure 2. Fig. 9 As shown, p72 protein after virus infection of cells can be fished up by 28H1 antibody.

[0086] The above results indicate that the antibody has a good affinity for the p72 protein in the natural state of virus infection, and can serve as a good tool for subsequent basic research on ASFVp72 protein and clinical diagnosis and prevention of African swine fever virus.

[0087] Example 4 PCR amplification and sequence determination of the variable region gene of p72 protein monoclonal antibody 28H1

[0088] The RNA of monoclonal antibody 28H1 hybridoma cells was extracted and reversed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). The variable region gene of the antibody was amplified by nested PCR. First, the above cDNA was used as a template to amplify the variable region gene of the antibody using the first round of mouse antibody IgG and κ light chain primers. Then, the first round product was used as a template to amplify the variable region gene of the antibody using the second round of mouse antibody IgG and κ light chain primers. The PCR reaction system was: 2×PhantaFlash Master Mix (Vazyme) 25μL, Primer1 and Primer2 1μL each, cDNA 1μL, ddH 2O was added to 50 μL. The reaction procedure was: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min. The primers for antibody variable region gene amplification refer to the literature (von Boehmer, L., Liu, C., Ackerman, S., Gitlin, AD, Wang, Q., Gazumyan, A., Nussenzweig, MC, 2016. Sequencing and cloning of antigen specific antibodies from mouse memory B cells. Nature protocols 11, 19081923.). After amplification, agarose gel electrophoresis was performed. The heavy chain and kappa light chain variable region gene size of monoclonal antibody 28H1 was about 300 bp ( Fig.10 ), the target fragment was recovered by gel excision, and the recovered target fragment was inserted into the pMD 18T blunt-end vector for sequence determination.

[0089] The sequencing results were compared and analyzed on the NCBI IGBLAST website, and the sequencing results confirmed that the amplified sequences were DNA of the heavy chain variable region and light chain variable region of the monoclonal antibody. Specifically, the amino acid sequence of the heavy chain variable region of the mouse anti-ASFV p72 protein monoclonal antibody 28H1 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the mouse anti-ASFV p72 protein monoclonal antibody 28H1 is shown in SEQ ID NO: 2. The DNA sequence encoding the heavy chain variable region of the mouse anti-ASFV p72 protein monoclonal antibody 28H1 is shown in SEQ ID NO: 3; the DNA sequence encoding the light chain variable region of the mouse anti-ASFV p72 protein monoclonal antibody 28H1 is shown in SEQ ID NO: 4.

[0090] SEQ ID NO: 1:

[0091] QVQLQQSGPELVRPGVSVKISCKGSGYTFTEYPIHWVKQSHAKSLEWIGIISTKYNNAG YNQKIKGKATLTVDKSSSTAYMELARLTSEDSAIYHCARNGGYLGDYWGQGTSVIVSS;

[0092] SEQ ID NO: 2:

[0093] DMVMTQSQKVMSTSVGDRVSVTCKADQNMGINVAWYQQKFGQSPQALIYSAPYRDS GVPDRFTGSGSGTDFTLTISDVQSEDLADYFCQRYNAFPYTFGGGTKLEIK;

[0094] SEQ ID NO: 3:

[0095] CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCCGGCTACACATTCACTGAATATCCTATACACTGGGTGAAGCAGAGTCATGCTAAGAGTCTAGAGTGGATTGGAATTATTAGTACTAAATATAACAATGCAGGATACAACCAGAAGATTAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTATATGGAGCTTGCCAGACTGACATCTGAGGATTCTGCCATCTATCACTGTGCAAGAAATGGTGGATATCTTGGCGATTACTGGGGTCAAGGAACCTCAGTCATCGTCTCCTCA;

[0096] SEQ ID NO: 4:

[0097] GACATGGTGATGACCCAGTCTCAAAAAGTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCGATCAGAATATGGGTATTAATGTAGCCTGGTATCAACAGAAATTTGGGCAATCTCCTCAAGCACTGATTTACTCGGCACCTTACCGGGACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCGATGTGCAGTCTGAAGACTTGGCAGACTATTTCTGTCAGCGCTATAACGCCTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。

[0098] The amino acid sequences of CDR1, CDR2 and CDR3 in the variable regions of the heavy and light chains of monoclonal antibody 28H1 are shown in Table 3 below.

[0099] Table 3 Amino acid sequences of CDR1, CDR2 and CDR3 of the heavy and light chain variable regions of monoclonal antibody 28H1

[0100] CDR1 CDR2 CDR3 Heavy chain oeLh IISTKYNNAGYNQKIKG NGGYLGDY Light chain KADQNMGINVA SAPYRDS QRYNAFPYT

[0101] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An anti-African swine fever virus p72 protein monoclonal antibody 28H1, characterized in that: The heavy chain variable region of the monoclonal antibody 28H1 includes a CDR1 with an amino acid sequence of EYPIH, a CDR2 with an amino acid sequence of IISTKYNNAGYNQKIKG, and a CDR3 with an amino acid sequence of NGGYLGDY; the light chain variable region of the monoclonal antibody 28H1 includes a CDR1 with an amino acid sequence of KADQNMGINVA, a CDR2 with an amino acid sequence of SAPYRDS, and a CDR3 with an amino acid sequence of QRYNAFPYT.

2. The monoclonal antibody 28H1 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 28H1 is shown in SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the monoclonal antibody 28H1 is shown in SEQ ID NO:

2.

3. The monoclonal antibody 28H1 according to claim 1, characterized in that The monoclonal antibody 28H1 generates a specific immune response with African swine fever virus or its p72 protein antigen.

4. The monoclonal antibody 28H1 according to claim 1, characterized in that The heavy chain subtype of the monoclonal antibody 28H1 is IgG1, and the light chain subtype is Kappa.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule is a nucleic acid molecule encoding the monoclonal antibody 28H1 according to any one of claims 1 to 4.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 5.

7. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 5 or the vector of claim 6.

8. Use of the monoclonal antibody 28H1 according to any one of claims 1 to 4, or the nucleic acid molecule according to claim 5, or the vector according to claim 6, or the host cell according to claim 7 in the preparation of a detection product for African swine fever virus or its p72 protein.

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

10. A product for detecting African swine fever virus or its p72 protein antigen, characterized in that: The product comprises the monoclonal antibody 28H1 according to any one of claims 1 to 4, or the nucleic acid molecule according to claim 5, or the vector according to claim 6, or the host cell according to claim 7.

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