African swine fever h240r epitope peptide, monoclonal antibody and application thereof

Abstract

The application discloses an African swine fever H240R epitope peptide, a monoclonal antibody and application thereof, and relates to the technical field of molecular immunology. The amino acid sequence of the African swine fever H240R epitope peptide is shown as SEQ ID NO. 19 or SEQ ID NO. 20. The monoclonal antibody is monoclonal antibody 11C1A6 or monoclonal antibody 38A3B2; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 11C1A6 are shown as SEQ ID NO. 9-10, respectively; the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 38A3B2 are shown as SEQ ID NO. 11-12, respectively. The African swine fever H240R epitope peptide is identified, and the monoclonal antibody which is specifically combined with the African swine fever H240R protein is prepared, thereby laying a foundation for preparing an African swine fever polypeptide vaccine and a detection kit.

Description

Technical Field

[0001] This invention relates to the field of molecular immunology technology, and in particular to African swine fever H240R epitope peptide, monoclonal antibody and their applications. Background Technology

[0002] African swine fever virus (ASFV) is the sole member of the Asfarviridae family and the only known DNA arbovirus (used to refer to any virus transmitted through arthropods). Although ASFV shares the same structure, genome, and replication characteristics as other NCLDVs, it differs in that it has a multilayered structure and an icosahedral morphology. Intracellularly, ASFV has a nucleoid containing its genome (first layer), surrounded by a thick protein layer (second layer), called the nucleocapsid (second layer), which is further encased by an endolithic membrane (third layer) and an icosahedral protein capsid (fourth layer); these four layers contain more than 50 proteins. When ASFV buds from the plasma membrane, it acquires an outer membrane (fifth layer). Significant gaps in knowledge regarding the composition, location, and structure of infectious viral particles, as well as the identification of viral proteins capable of inducing a protective immune response in pigs, have hindered vaccine development.

[0003] Based on proteomics analysis, protein abundance levels in ASFV particles, and similarity between cryo-electron microscopy images and predicted structural features of target proteins (including protein sequence, secondary structure, and topology), a pentamer protein (H240R) and three secondary capsid proteins (p17, p49, p17, p49, and H240R) were identified. Each icosahedral asymmetric unit of the outer capsid contains 46 pseudo-hexahedral capsids, of which 6 are in the pentamer (a, b, c, d, e, f) and 40 are in the trimer (a, b, c, d, e, f, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y, Z, A′, B′, C′, D′, E′, F′, G′, H′, I′, J′, K′, L′, M′, and N′). The H240R protein and secondary capsid proteins (p17, p49, and M1249L) form a complex network immediately below the outer capsid, stabilizing the entire capsid.

[0004] BLAST alignment of open reading frames of penton protein sequences was performed on all ASFV viral strains, identifying the homolog H240R with 37.7% sequence similarity. This is an uncharacterized but crucial viral particle protein. H240R is 240 residues long, β-rich, and features a coil fold and an N-terminal extension of approximately 70 amino acids, further supporting the identifying function of the penton in ASFV. A low-density [structure / feature] was observed below the penton. The H240R protein has a lantern-like structure that connects five adjacent cells, the inner membrane, and five adjacent capsids. It was previously speculated that the H240R protein plays a role in vertex assembly. Currently, research on the ASFV H240R protein is scarce. Therefore, it is necessary to recombinantly express the H240R protein and prepare its specific monoclonal antibody, and then use bioinformatics and peptide scanning techniques to screen and identify the H240R protein. Summary of the Invention

[0005] The purpose of this invention is to provide African swine fever H240R epitope peptides, monoclonal antibodies, and their applications to solve the problems existing in the prior art. This invention identifies African swine fever H240R epitope peptides and prepares monoclonal antibodies that specifically bind to African swine fever H240R proteins, laying the foundation for the preparation of African swine fever polypeptide vaccines and detection kits.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides an African swine fever H240R epitope peptide, the amino acid sequence of which is shown in SEQ ID NO.19 or SEQ ID NO.20.

[0008] The present invention also provides a gene encoding the above-mentioned African swine fever H240R epitope peptide.

[0009] The present invention also provides a biological material comprising the above-mentioned coding gene, wherein the biological material comprises a recombinant expression vector or a recombinant microbial strain.

[0010] The present invention also provides the application of the above-mentioned African swine fever H240R epitope peptide, encoding gene or biological material in the preparation of African swine fever polypeptide vaccine.

[0011] The present invention also provides an African swine fever polypeptide vaccine, comprising the above-mentioned African swine fever H240R epitope peptide.

[0012] The present invention also provides an African swine fever H240R homodimeric protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0013] The present invention also provides the application of the above-mentioned African swine fever H240R homodimer protein in the preparation of African swine fever H240R monoclonal antibody.

[0014] The present invention also provides a monoclonal antibody against African swine fever H240R, wherein the African swine fever H240R monoclonal antibody is monoclonal antibody 11C1A6 or monoclonal antibody 38A3B2.

[0015] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 11C1A6 is shown in SEQ ID NO.9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10.

[0016] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 38A3B2 is shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.12.

[0017] The present invention also provides the application of the above-mentioned anti-African swine fever H240R monoclonal antibody in the preparation of African swine fever detection kits.

[0018] The present invention also provides an African swine fever detection kit, comprising the above-mentioned anti-African swine fever H240R monoclonal antibody.

[0019] The present invention discloses the following technical effects:

[0020] This invention, through preliminary screening and truncation identification, yielded... 150 LSGPLD 155 and 170 HIQLP 175 This is the B-cell epitope region of H240R, which lays the foundation for the preparation of African swine fever peptide vaccines.

[0021] This invention optimizes the nucleic acid sequence of the H240R homodimeric protein to improve its expression in an insect baculovirus expression system, preparing virus-like particles of recombinant ASFV H240 dimeric protein. Immunization of mice with these particles yields monoclonal antibodies that specifically bind to H240R. This invention provides a novel source of monoclonal antibodies for the preparation of African swine fever detection kits. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Prediction of transmembrane regions for the H240R protein;

[0024] Figure 2 The amino acid sequence of the ASFV H240R homodimer protein;

[0025] Figure 3 Codon optimization for the eukaryotically expressed H240R protein; where A is codon preference analysis; B is codon fitness index (CAI) analysis after optimization; C is GC content analysis;

[0026] Figure 4 To obtain the target gene for the ASFV H240R homodimer;

[0027] Figure 5 The results of PCR for recombinant plasmid pFastBacI-H240R bacterial culture;

[0028] Figure 6 Results of PCR performed on white monoclonal bacteria selected for blue-white screening;

[0029] Figure 7 The results of Western blot identification of eukaryotic H240R protein expression are as follows: A is the Western blot identification of H240R protein expression in third-generation virus P3; B is the Western blot identification of H240R protein expression in sf21 cells; C is the analysis of the reaction between recombinant H240R protein and positive serum from recovered pigs.

[0030] Figure 8 Results of IFA identification of eukaryotic H240R protein expression;

[0031] Figure 9 The results show the purification and identification of the eukaryotic H240R protein; where A is the structure of H240R after preliminary purification by nickel affinity chromatography; B is the SDS-PAGE identification of the H240R protein obtained by SP cation exchange chromatography purification; and C is the Western blot identification of the eluents of each component after SP cation exchange chromatography purification.

[0032] Figure 10 The reaction of purified eukaryotic H240R dimer recombinant protein with positive serum;

[0033] Figure 11 It is a virus-like particle of H240R;

[0034] Figure 12 Morphology of Sp2 / 0 myeloma cells under an optical microscope;

[0035] Figure 13The results are from the determination of the ascites titer of ASFV H240R monoclonal antibody;

[0036] Figure 14 The reaction of monoclonal antibodies with recombinant H240R protein expressed in eukaryotic cells was detected by Western blot and IFA. Specifically, A shows the reaction of monoclonal antibody 11C1A6 with recombinant H240R protein identified by Western blot; B shows the reaction of positive serum with recombinant H240R protein identified by Western blot; C shows the reaction of monoclonal antibody 38A3B2 with recombinant H240R protein identified by Western blot; D shows the reaction of monoclonal antibody 11C1A6 with H240R protein expressed in sf21 cells identified by IFA; E shows the reaction of positive serum with H240R protein expressed in sf21 cells identified by IFA; and F shows the reaction of monoclonal antibody 38A3B2 with H240R protein expressed in sf21 cells identified by IFA.

[0037] Figure 15 The results show the amplification of the heavy chain variable region and light chain variable region of the H240R monoclonal antibody.

[0038] Figure 16 To preliminarily screen B-cell epitopes of H240R using indirect ELISA; where A represents the reaction with monoclonal antibody 11C1; and B represents the reaction with monoclonal antibody 38A3.

[0039] Figure 17 To preliminarily screen B-cell epitopes of H240R protein using Dot-ELISA; where A represents the reaction with monoclonal antibody 11C1; and B represents the reaction with monoclonal antibody 38A3.

[0040] Figure 18 The results show the truncated identification of the H240R protein B-cell epitope; where A is the indirect ELISA test of the reactivity of the six-segment short peptide with anti-H240R monoclonal antibody 11C1A6; B is the indirect ELISA test of the reactivity of the six-segment short peptide with anti-H240R monoclonal antibody 38A3B2; C is the Dot-ELISA test of the reactivity of the six-segment short peptide with anti-H240R monoclonal antibody 11C1A6; and D is the Dot-ELISA test of the reactivity of the six-segment short peptide with anti-H240R monoclonal antibody 38A3B2.

[0041] Figure 19 The spatial structure of the African swine fever H240R epitope peptide. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0046] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0047] Example 1: Amino acid sequence analysis, optimization, and gene synthesis of ASFV H240R protein

[0048] Based on the sequence of the ASFV China / 2018 / AnhuiXCGQ isolate (GeneBank accession number: MK128995.1), bioinformatics analysis was performed on the sequence of the H240R gene (protein id: AYW34090.1), encoding ASFV H240R protein (155365-156090 bp). The transmembrane region of the H240R protein was analyzed based on its amino acid sequence using TMHMM Server v.2.0. Figure 1 As shown, amino acids 1-241 of the H240R protein are located outside the cell membrane, therefore, the full-length H240R sequence needs to be expressed. The amino acid sequence of the H240R protein was modified to express it as a homodimer, thereby enhancing its immunogenicity and immunoreactivity. For example... Figure 2As shown, the amino acid sequence of the H240R protein homodimer is GP67-H240R-Linker-H240R-6×his (amino acid sequence see SEQ ID NO.1). Adding a gp67 signal peptide to the 5' end of the H240R homodimer sequence enables secretory expression, which is beneficial for protein folding, glycosylation, phosphorylation, and other processing, and also facilitates the acquisition of high-purity target protein. Simultaneously, adding a 6×his tag to the 3' end of the H240R homodimer sequence facilitates affinity purification of the protein.

[0049] like Figure 3 As shown, the nucleic acid sequence of the H240R homodimeric protein was optimized using codon optimization software from Shanghai Sangon Biotech Co., Ltd., resulting in better expression in the insect baculovirus expression system. The optimized CAI was 0.98. Figure 3 (B) The average GC content was adjusted to 54.33% ( Figure 3 (C). The optimized nucleic acid sequence of the ASFV H240R homodimer protein is shown in SEQ ID NO.2, with a full length of 1626 bp. Analysis of its physicochemical properties using ProtParam bioinformatics software revealed that the ASFV H240R homodimer protein has an isoelectric point (pI) of 9.45 and a molecular weight of 625 kDa.

[0050] The optimized sequence was ligated into the pUC57 vector at Shanghai Sangon Biotech Co., Ltd. to synthesize the target gene pUC57-H240R.

[0051] SEQ ID NO.1:

[0052] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAEFMAANIIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPFMIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPNQVELLPVNNKYKFTYAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCKNNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSKGGGGSGGGGSGGGGSMAANIIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPFMIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPNQVELLPVNNKYKFTYAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCKNNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSKHHHHHH。

[0053] SEQ ID NO.2:

[0054]

[0055] Example 2: Construction of the pFastBacI-H240R protein recombinant vector

[0056] Primers for amplifying the target gene were designed based on the nucleotide sequence of the H240R homodimeric protein, and two restriction enzyme sites, BamHI and XbaI, were introduced. The designed primers are shown in Table 1. Figure 4 As shown, 1% agarose gel electrophoresis results indicated that a 1626 bp H240R homodimer gene was amplified from the synthesized pUC57-H240R. After double enzyme digestion, DNA ligation, and transformation of the ligation product into DH5α competent cells (using pFastBacI as the vector, provided by the Animal Immunology Laboratory of Henan Academy of Agricultural Sciences), bacterial PCR was performed using M13 universal primers. The agarose gel electrophoresis results are shown below. Figure 5 As shown, the size of a positive clone is the target gene length plus approximately 2300 bp, therefore the size of the H240R protein positive clone pFastBacI-H240R is 3926 bp. Two positive clones of the correct size, pFastBacI-H240R-9 and pFastBacI-H240R-15, were randomly selected for bacterial sequencing. Sequence alignment using Meglin showed that both pFastBacI-H240R-9 and pFastBacI-H240R-15 were identical to the H240R homodimer sequence. Recombinant plasmids were randomly selected from pFastBacI-H240R-9 to prepare recombinant stickymids.

[0057] Table 1 Primers for amplifying the target gene of the H240R homodimer.

[0058]

[0059] Note: Italics indicate enzyme cleavage sites.

[0060] Example 3: Preparation of pFastBacI-H240R recombinant clay particles

[0061] Add 50 μL of the recombinant strain pFastBacI-H240R constructed in Example 2 to 5 mL of solution containing Amp +The cells were cultured overnight at 37°C in a shaker at 220 rpm in LB medium. The next day, the cells were collected by centrifugation at 12,000 rpm for 1 min. The recombinant plasmid pFastBacI-H240R was extracted using the OMEGA plasmid miniprep kit, and its concentration was determined. The recombinant plasmid pFastBacI-H240R was transformed into DH10Bac competent cells. Large, round, white single colonies were picked and transferred to 1 mL of LB medium containing three antibiotics at final concentrations of 50 μg / mL Kana, 50 μg / mL Tet, and 7 μg / mL Gen. The cells were then cultured overnight in a shaker at 37°C in 200 rpm. After the bacterial culture became turbid, the bacterial culture was used for PCR and sequencing identification. The correctly sequenced recombinant plasmids were then inoculated at a ratio of 1:100 into LB medium containing triple antibodies (KANA, Gen, and Tet) and incubated at 37°C and 200 rpm for at least 12 hours. Recombinant Bacmids were extracted using the OMEGA endo-free plasmid mini extraction kit, and the concentration of Bacmids was determined using a microdroplet UV spectrophotometer. The Bacmids were aliquoted at 10 μL / tube into sterile 1.5 mL centrifuge tubes and stored at -20°C for subsequent experiments. Note that Bacmids are large and unstable, and prone to breakage; therefore, repeated freeze-thaw cycles should be avoided.

[0062] The results are as follows Figure 6 As shown, of the seven clones of pFastBacI-H240R (DH10Bac10), all except clone 4 were positive, with clone 7 being a strongly positive clone. The sequencing results of clone 7, when aligned with Meglin, showed complete consistency with the original synthesized sequence, allowing for the extraction of granules for further transfection.

[0063] Example 4: Expression of ASFV H240R dimer protein in a eukaryotic baculovirus expression system

[0064] Following the standard operating procedures for the baculovirus expression system in the Bac-to-Bac operating system, after harvesting the third-generation virus (P3) containing the ASFV H240R protein, the virus was amplified in large quantities, and recombinant H240R protein was harvested. Western blot analysis was performed on the cell culture supernatant, and the results are as follows: Figure 7 As shown. Figure 7Figure A shows a Western blot analysis of H240R third-generation virus P3, using a murine HRP-labeled his monoclonal antibody. The image shows a single specific band at approximately 63 kDa, consistent with the predicted size of the H240R dimer protein. Further Western blot analysis of the harvested 300 mL of amplified H240R protein confirmed successful expression of the ASFV H240R dimer protein in sf21 cells. Figure 7 (B) and used positive serum from ASFV-recovered pigs as the primary antibody and goat anti-pig IgG as the secondary antibody to identify the reactivity of H240R with the positive serum, such as Figure 7 The results from the C-cell assay showed that the H240R dimer protein reacted well with the positive serum of recovered pigs. IFA experiments showed that the H240R dimer protein was well expressed in the insect baculovirus expression system, such as... Figure 8 As shown.

[0065] Example 5: Purification of recombinant ASFV H240 dimer protein

[0066] The 6×his fusion tag of the eukaryotic ASFV H240R protein was used for initial purification. This tag binds to a nickel affinity chromatography column and dissociates under the action of imidazole. The imidazole concentration in the washing solution for other proteins was 10 mM, and the concentration of imidazole in the elution solution for the target protein was 50 mM. Further purification of the product obtained from affinity chromatography was performed using an SP cation exchange column, yielding H240R protein with a purity of 90%. Figure 9 As shown in Figure A, the SDS-PAGE results of Ni affinity chromatography purification show the target protein at approximately 63 kDa, but several other bands are still present. Further purification of the Ni affinity-purified protein was performed using SP cation exchange chromatography. Figure 9 As shown in Figure B, the collected SP purified flow-through solution was concentrated using a 10 kDa ultrafiltration tube to obtain a single target band at 63 kDa. Figure 9 As shown in Figure C, the proteins collected at each stage of SP purification were identified using his monoclonal antibody as the primary antibody in Western blot analysis. The results showed that the H240R protein was present in the flow-through solution. Figure 10 As shown, the purified H240R dimer protein specifically reacted with the positive serum of recovered pigs, and the specific reaction band was located at approximately 63 kDa. The concentration of H240R dimer protein, as determined by the BCA protein concentration assay kit, was 1.47 mg / mL. The purified H240R protein solution was observed using a JEM-1400 (80 kV) transmission electron microscope. Figure 11 As shown, H240R protein particles aggregate into virus-like particles (VLPs) of about 9 nm in size.

[0067] Example 6: Animal Immunization and Hyperimmunization of Recombinant ASFV H240 Dimeric Protein

[0068] Following a standard immunization protocol, as shown in Table 2, three Balb / c mice were immunized on days 0, 14, and 21. A booster immunization was administered on day 35 to the mouse with the highest titer on day 21. For the primary immunization, 25 μg of ASFV H240R expressed by eukaryotic baculovirus was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple points on the back of the neck of each mouse. For the second and third primary immunizations, 25 μg of ASFV H240R protein was emulsified with Freund's incomplete adjuvant and administered as a booster immunization 3 days before cell fusion, with 25 μg of ASFV H240R protein injected intraperitoneally without adjuvant.

[0069] Table 2. Immunization regimens for mice

[0070]

[0071] Example 7: Culture and fusion of myeloma cells

[0072] Through cell resuscitation and passage culture of myeloma cells, the cells were cultured until four flasks of T75 cells were confluent, and the cells were in good condition. Figure 12 As shown, the cells adhered to the bottom of the flask, arranged neatly in a semi-dense distribution, round and translucent, uniform in size, and with clear edges. Mouse spleen cells extracted after immunization in Example 6 were fused with sp2 / 0 hybridoma cells using the PEG method. The suspended cells were dispersed into 10 96-well cell culture plates, with 250 μL of cell suspension added to each well. Small cell clusters could be observed under a microscope after 4-5 days of culture, and the hybridoma cell supernatant was analyzed after 9-12 days. Five monoclonal cell lines that reacted well with the H240R protein were screened using an indirect ELISA method and named 11C1 and 38A3, respectively.

[0073] Example 8: Identification of Monoclonal Antibodies

[0074] The ascites titers of the two positive monoclonal cell lines against ASFV H240R protein obtained in Example 7, namely 11C1 and 38A3, were determined. Figure 13 As shown, the titers of monoclonal antibodies 11C1A6 and 38A3B2 were 1:108 and 1:109, respectively. Isotype determination of the two anti-ASFV H240R monoclonal antibodies using an isotype identification kit revealed that both 11C1A6 and 38A3B2 had IgG1 heavy chains and Kappa light chains. The reaction between ascites fluid containing anti-H240R monoclonal antibodies and recombinant H240R protein was detected using Western blot and IFA. Results are shown below. Figure 14As shown, both monoclonal antibodies against H240R reacted with the H240R protein, with specific bands around 63 kDa. Figure 14 In section A, monoclonal antibody 11C1A6 reacts with H240R. Figure 14 In section C, monoclonal antibody 38A3B2 reacts with H240R. Figure 14 E represents the reaction between positive serum and H240R. Figure 14 In the middle three cells, B, D, and F represent monoclonal antibodies 11C1A6 and 38A3B2, respectively, and positive serum, all of which reacted with the H240R protein expressed in sf21 cells.

[0075] RNA was extracted from the H240R monoclonal antibody, and the heavy chain variable region (VH) and light chain variable region (VL) of the H240R monoclonal antibody were amplified using conventional PCR methods. Figure 15 As shown, the first five lanes contain the heavy chain variable region gene of monoclonal antibody-derived ASFV H240R protein from cell 11C1A6 and the light chain variable region gene amplified by four different primer pairs. The next five lanes contain the heavy chain variable region gene of monoclonal antibody-derived ASFV H240R protein from cell 38A3B2 and the light chain variable region gene amplified by four different primer pairs. The last lane contains a 5000bp nucleic acid marker. The figure shows that the target gene size for both the heavy and light chain variable regions is approximately 300bp.

[0076] Monoclonal antibody against ASFV H240R protein

[0077] The 11C1A6 heavy chain variable region nucleic acid sequence (SEQ ID NO.5) is 353 bp (5'-3').

[0078] TTTTGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAGTAAGCAAACGGGTGGGCCGTACAGTAATAAATGCCAGTGTCTTCAGCTTTTAAGCTGTTCATTTGCAGGTAGACACTACTTTTGGAATCATCTCTTGAGATGGTGAACCTCCCTTTCACAGACTCAGCATAGTATGTTGC ATGATTATTAGCTTTGCTTCTAATTTCAGCAACCCACTCAAGCCCCTTCTCTGGAGACTGGCGGACCCAGTCCATCCAGGCGTCACTAAAAGTGAATCCAGAGGCAGCACAAGAGAGTTTCATGGATCCTCCAGGTTGCACCAAGCTCCTCCAGACTCCTGCAGCTGCACCTCAA.

[0079] The nucleic acid sequence of the variable region of the light chain of 11C1A6 (SEQ ID NO.6) is: 220bp (5'-3')

[0080] TCCGTTTCAGCTCCAGCTTGGTCCCCCCTCCGAACGTGTACGGAACATGTGAACCTTGAAAGCAGTAATAAACTCCCAGATCCTCAGCCTCCACTCTGCTGATCTTGAGTGTGAAATCTGTCCCTGATCCACTGCCACTGAACCTGTCTGGGACCCCAGAAAATCGGTTGGAAACTTTGTAGATCAGGAGCTTTGGAGACTGGGTGAGCTCAATGTCAAA。

[0081] The nucleic acid sequence of the variable region of the heavy chain of 38A3B2 (SEQ ID NO.7) is: 360bp (5'-3')

[0082] TTGAGGTGCAGCTGCAGGAGTCAGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGACTCTCTCCTGTGTAGCCTCAGGATTCGATTTAAGTAGATACTGGATGAGTTGGGCTCGGCAGGCTCCAGGGAAAGGGCAGGAATGGATTGGAGAAATTAATCCAGGAAGCAGTACGATAAACTATGCGCCGTCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACTGGGCCTTTACGGCTGGTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAAAAA。

[0083] The nucleic acid sequence of the variable region of the light chain of 38A3B2 (SEQ ID NO.8) is: 327bp (5'-3')

[0084] TTGACATTGAGCTCACCCAGTCTCCAGCCATCACAGCTGCATCTCTGGGGCAAAAGGTCACCATCACCTGCAGTGCCAGTTCAAGTGTAAGTTACATACACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAACCATGGATTTATGAAATATCCAAACTGGCT TCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCATTTATTACTGCCAGCAGTGGAGTTATCCTCTTATCACATTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGACAA.

[0085] The amino acid sequence of the H240R monoclonal antibody was obtained using the ORF finder online prediction software in NCBI:

[0086] The amino acid sequence of the variable region of the 11C1A6 heavy chain (SEQ ID NO.9) is as follows:

[0087] MEVQLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKA NNHATYYAESVKGRFTISRDDSKSSVYLQMNSLKAEDTGIYYCTAHPFAYWGQGTTVTVSS K.

[0088] The amino acid sequence of the 11C1A6 light chain variable region (SEQ ID NO.10) is as follows:

[0089] MFDIELTQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLELKR.

[0090] The amino acid sequence of the variable region of the 38A3B2 heavy chain (SEQ ID NO.11) is as follows:

[0091] SK.

[0092] The amino acid sequence of the 38A3B2 light chain variable region (SEQ ID NO.12) is as follows:

[0093] MDIELTQSPAITAASLGQKVTITCSASSSVSYIHWYQQKSGTSPKPWIYEISKLASGVPA RFSGSGSGTSYSLTISSMEAEDAAIYYCQQWSYPLITFGAGTKLELKRT.

[0094] The heavy chain variable region sequences and light chain variable region sequences of each monoclonal antibody are linked together using a flexible linker in the order VH-Linker-VL to form a single-chain antibody.

[0095] Example 9: Preliminary screening of B-cell epitopes of H240R protein

[0096] The results of the indirect ELISA reaction between the anti-H240R protein monoclonal antibody and 24 short peptides are as follows: Figure 16 As shown. Short peptide number 15 ( 141 QYLTPIFYDLSGPLD 155 ), its average OD 450 Value 1.353, short peptide No. 16 ( 151 SGPLDFPLDTLSVHV 165 ), its average OD 450 Value 0.697, short peptide No. 17 ( 161 LSVHVDILSNHIQLP 175 ), average OD 450 The value is 0.802, and it is similar to 11C1 (e.g.) Figure 16 A) undergoes a reaction. Short peptide number 15 ( 141 QYLTPIFYDLSGPLD 155 ) and 38A3 ( Figure 16 The average OD of reaction B) 450 The value is 1.455, short peptide number 16 ( 151 SGPLDFPLDTLSVHV 165 ) and 38A3 ( Figure 16 The average OD of reaction B) 450 Value 0.736, short peptide No. 17 ( 161 LSVHVDILSNHIQLP 175 ) and 38A3 ( Figure 16 In reaction B), the average OD 450 The value is 0.818. Therefore, peptides 15, 16, and 17 all showed strong reactions with the monoclonal antibody, suggesting that peptides 15, 16, and 17 are possible B-cell epitope regions of the H240R protein.

[0097] The H240R protein B-cell epitopes were initially screened using the Dot-ELISA method, such as... Figure 17 As shown. Short peptides 1-24 were spotted onto the NC membrane and incubated with two monoclonal antibodies. The results showed that short peptide 15 ( 141 QYLTPIFYDLSGPLD 155 ), 16 short peptide ( 151 SGPLDFPLDTLSVHV 165 ), 17 short peptide ( 161 LSVHVDILSNHIQLP 175 ) and 11C1( Figure 17 (A) and 38A3 ( Figure 17 Both B) monoclonal antibodies showed strong responses, a result consistent with that of indirect ELISA.

[0098] Example 10: Identification of truncated B-cell epitopes of the H240R protein

[0099] The short peptides 15, 16, and 17 identified in the initial screening of the H240R protein were combined together, i.e. 141 QYLTPIFYDLSGPLDTLSVHVDILSNHIQLP 175 The amino acid residues from positions 141 to 175 were divided into six segments using the overlapping peptide method, as shown in Table 3. To enable the short peptides to be coupled to the carrier protein BSA, cysteine ​​(Cys C) was added to the N-terminus of each of the six synthesized truncated peptides. After coupling the short peptides and BSA using the SMCC method, the reactivity of these six short peptides with two anti-H240R monoclonal antibodies was identified using indirect ELISA. Figure 18 From A and B, we know that only Truncation-2 ( 146 IFYDLSGPLD 155 ) and Truncation-6 ( 166 DILSNHIQLP 175 It reacted with two monoclonal antibodies against H240R, OD 450 The value is 0.964. For example... Figure 18 As shown in C and D, the Dot-ELISA results are consistent with the indirect ELISA results. The secondary structure properties of the merged sequences were analyzed using Protean plates in DNAstar software, where only... 150 LSGPLD 155 The antigenic index of residue (SEQ ID NO.19) is positive, consistent with the results of indirect ELISA and Dot-ELISA. Although the remaining peptides exhibit folded, helical, and turn structures, their antigenicity and surface accessibility are all negative. 166DILSNHIQLP 175 A β-sheet structure exists. Therefore, combining experimental and predicted results, it is concluded that... 150 LSGPLD 155 and 170 HIQLP 175 (SEQ ID NO.20) represents the B-cell epitope region of H240R. The spatial structure of the African swine fever H240R epitope peptide is shown below. Figure 19 As shown.

[0100] Table 3 shows the truncation of peptides initially screened by H240R.

[0101]

[0102]

[0103] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An anti-African swine fever H240R monoclonal antibody, characterized in that, The African swine fever H240R monoclonal antibody is monoclonal antibody 38A3B2. The amino acid sequence of the heavy chain variable region of the monoclonal antibody 38A3B2 is shown as SEQ ID NO. 11, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

12.

2. Use of the anti-African swine fever H240R monoclonal antibody of claim 1 in the preparation of an African swine fever detection kit.

3. An African swine fever detection kit, characterized by, The anti-African swine fever H240R monoclonal antibody of claim 1.

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