African swine fever virus CD2V epitope peptide and application thereof, and anti-african swine fever virus CD2V monoclonal antibody

Abstract

The present application relates to African swine fever virus CD2V epitope peptide and application thereof, and anti-African swine fever virus CD2V monoclonal antibody. The present application uses recombinant ASFV CD2V as immunogen to immunize animals, and 5 strains of anti-ASFV monoclonal antibodies are prepared. The extracellular region of ASFV CD2V protein is truncated into 18 short peptides by using peptide scanning method, i.e. overlapping polypeptide method, and the 18 short peptides are respectively reacted with the 5 strains of anti-ASFV monoclonal antibodies to identify 5 epitope peptides, and the amino acid sequences of the 5 epitope peptides are shown as SEQ ID NO:1-5. The epitope peptides of the present application can be used as immunogen to stimulate animals to produce anti-ASFV serum or anti-ASFV monoclonal antibody; can be used for preparing ASFV vaccine; can be used for preparing African swine fever virus immunological detection reagent or kit, and provides new antigen target for ASFV vaccine design, antibody research and development, and antibody detection kit research.

Description

Technical Field

[0001] This invention belongs to the field of molecular immunology technology, specifically relating to the African swine fever virus CD2V epitope peptide and its application, and anti-African swine fever virus CD2V monoclonal antibody. Background Technology

[0002] African swine fever virus (ASFV) is the pathogen of African swine fever (ASF), a highly pathogenic and fatal disease in pigs. ASFV is a large, double-stranded DNA virus (140-170 kDa) and the only member of the Viridae family. Since 2007, ASF has caused devastating outbreaks and has been persistent in the Caucasus region, the Russian Federation, the Baltic states, Eastern European countries, and China. This disease poses a serious threat to the global pig industry. Although protection against homologous viral challenge has been found, there is currently no commercially available vaccine for ASF. The lack of knowledge about ASFV anti-gene-induced protective immunity and the diversity of these protective antigens in nature has hindered the development of ASF vaccines and the progress of disease control.

[0003] Currently, the protective immunity against ASFV remains unclear. As with most viral infections, protective proteins appear important for humoral infection and cellular immune responses. However, whether the passive transfer of anti-ASFV antibodies has a protective effect, and its mechanism of action, remains unclear. There are reports on ASFV neutralizing antibodies, but their cross-neutralizing activity in vitro is not associated with cross-protection against ASFV in pigs. ASFV protective immunity may be serotype-specific; for example, cross-protection occurs in the hemoadsorption-inhibiting (HAI) serum group when the virus is present, while no cross-protection is observed in the control group. Interestingly, anti-ASFV monocyte infection-inhibiting (M-II) antibodies inhibit ASFV replication in macrophage cultures, but only to some extent inhibit in vivo cross-protective immunity associated with homologous ASFV strains.

[0004] Studies have shown that cellular immune responses play a crucial role in protective immunity against ASFV. The depletion of porcine lymphocytes indicates that cytotoxic CD8+ lymphocytes are an important pathway for clearing ASFV and generating protection, and the protective effect of proteins is related to ASFV strain-specific CD8+ T cell responses. Furthermore, in animals immunized with DNA vaccines, no anti-ASFV antibodies were detected upon challenge, further demonstrating the role of cellular immunity in protection. Therefore, some viral proteins can induce robust protective immunity in pigs.

[0005] The ASFV CD2v (EP402R) and c-type lectin (EP153R) proteins exhibit serological specificity. ASFV CD2v is the only known viral homolog of cellular CD2, a T-cell protein involved in the co-regulation of cell activation. CD2v is the hemagglutinin of ASFV and participates in protective immunization. Immunization of pigs with CD2v yields antibodies against HAI and M-II and provides partial protection against challenge from virulent syngeneic strains. Subunit ASF vaccines constructed using CD2v expression offer partial protection. ASFV chimeric virus vaccines also demonstrate the important role of CD2v in protective immunization; homologous CD2v and the adjacent c-type lectin protein are essential for preventing homologous ASFV infection. Therefore, CD2v and c-type lectin proteins may be important protective antigens of ASFV and should be targets for vaccine design and development. However, the viral CD2V protein domains and epitopes associated with host protective responses remain to be determined. Summary of the Invention

[0006] The present invention aims to provide an African swine fever virus (ASFV) CD2V epitope peptide, offering a novel antigenic target for ASFV vaccine design, antibody development, and antibody detection kit development. A second objective is to provide a nucleic acid molecule encoding the aforementioned epitope peptide. A third objective is to provide an expression cassette, recombinant vector, recombinant cell, or recombinant bacteria containing the aforementioned nucleic acid molecule. A fourth objective is to provide applications of the aforementioned epitope peptide, nucleic acid molecule, expression cassette, recombinant vector, recombinant cell, or recombinant bacteria. A fifth objective is to provide an anti-ASFV CD2V monoclonal antibody that recognizes the aforementioned epitope peptide. A sixth objective is to provide a nucleic acid molecule encoding the aforementioned monoclonal antibody.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] This invention provides an African swine fever virus CD2V epitope peptide, the amino acid sequence of which is shown in any one of SEQ ID NO:1-5.

[0009] This invention constructs a prokaryotic expression vector for ASFV CD2V, introduces it into *E. coli* for induced expression, and purifies the recombinant ASFV CD2V soluble protein using Ni column affinity chromatography. Animal immunization with the recombinant ASFV CD2V yields five anti-ASFV monoclonal antibodies. Finally, using a peptide scanning method (overlapping peptide method), the extracellular region of the ASFV CD2V protein is truncated into 18 short peptides. These 18 short peptides are reacted with the five anti-ASFV monoclonal antibodies, identifying five epitope peptides with amino acid sequences shown in SEQ ID NO: 1-5. The truncated peptide assay confirms that the three epitopes shown in SEQ ID NO: 1-3 represent the minimum length of B-cell epitopes for the ASFV CD2V protein. These results indicate that the newly discovered antigenic epitopes on ASFV CD2V provided by this invention possess strong immunoreactivity and immunogenicity, offering new antigenic targets for ASFV vaccine design, antibody development, and antibody detection kit development.

[0010] The present invention also provides a nucleic acid molecule encoding the above-mentioned African swine fever virus CD2V epitope peptide.

[0011] Due to the degeneracy of codons, based on the amino acid sequence of the epitope peptide described above, there can be multiple nucleotide sequences capable of encoding the specific antigenic epitope described in this invention. The linear epitope peptide described in this invention can be prepared artificially (e.g., using solid-phase polypeptide synthesis methods) or through genetic engineering methods; wherein the genetic engineering method involves operatively linking the encoding nucleic acid molecule of the epitope peptide to an expression vector, transforming it into a host cell to induce expression, collecting the expressed protein, and purifying it.

[0012] The present invention also provides expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria containing the above-mentioned nucleic acid molecules.

[0013] Specifically, the recombinant vector is selected from prokaryotic or eukaryotic expression vectors. Further, the recombinant vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. The expression system is a bacterial, yeast, filamentous fungal, mammalian cell, insect cell, plant cell, or cell-free expression system.

[0014] The present invention can also further modify or optimize the nucleic acid molecule encoding the epitope peptide to improve its expression level or expression efficiency in host cells. These modification or optimization methods are well known to those skilled in the art.

[0015] The epitope peptides, nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria described in this invention are all beneficial for the prevention and / or treatment of African swine fever (ASF). Therefore, this invention also provides the application of the above-mentioned epitope peptides, nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria in the preparation of drugs for the prevention and / or treatment of African swine fever virus.

[0016] Preferably, the drug is a vaccine. The vaccine includes one or more epitope peptides shown in SEQ ID NO:1-5.

[0017] Preferably, the drug is anti-ASFV serum or anti-ASFV monoclonal antibody. The anti-ASFV serum or anti-ASFV monoclonal antibody is prepared using one or more of the epitope peptides shown in SEQ ID NO:1-5 as an immunogen.

[0018] This invention also provides the application of the above-mentioned epitope peptides, nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria in the preparation of immunodetection reagents or kits for African swine fever.

[0019] Those skilled in the art will understand that the kit provided by the present invention can immobilize epitope peptides on an ELISA plate for the detection of ASFV-specific antibodies (such as serum antibodies), or can use one or more of the epitope peptides shown in SEQ ID NO:1-5 as immunogens to prepare anti-ASFV antibodies and coat them on an ELISA plate for the detection of ASFV CD2V protein.

[0020] This invention provides a monoclonal antibody against African swine fever virus CD2V that recognizes the above-mentioned epitope peptide, wherein the monoclonal antibody is any one of the following:

[0021] 1) Containing the amino acid sequences VHCDR1, VHCDR2, and VHCDR3 as shown in SEQ ID NO:7-9, and the amino acid sequences VLCDR1, VLCDR2, and VLCDR3 as shown in SEQ ID NO:10-12; 2) Containing the amino acid sequences VHCDR1, VHCDR2, and VHCDR3 as shown in SEQ ID NO:15-17, and the amino acid sequences VLCDR1, VLCDR2, and VLCDR3 as shown in SEQ ID NO:18-20; 3) Containing the amino acid sequences VHCDR1, VHCDR2, and VHCDR3 as shown in SEQ ID NO:23-25, and the amino acid sequences VLCDR1, VLCDR2, and VLCDR3 as shown in SEQ ID NO:26-28; 4) Containing the amino acid sequences VHCDR1, VHCDR2, and VHCDR3 as shown in SEQ ID NO:31-33, and the amino acid sequences VLCDR1, VLCDR2, and VLCDR3 as shown in SEQ ID NO:34-36;

[0022] It contains the amino acid sequences VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO:39-41, and the amino acid sequences VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO:42-44.

[0023] Preferably, the monoclonal antibody is any one of the following: 1) comprising a heavy chain variable region with an amino acid sequence as described in SEQ ID NO:13 and a light chain variable region with an amino acid sequence as described in SEQ ID NO:14; 2) comprising a heavy chain variable region with an amino acid sequence as described in SEQ ID NO:21 and a light chain variable region with an amino acid sequence as described in SEQ ID NO:22; 3) comprising a heavy chain variable region with an amino acid sequence as described in SEQ ID NO:29 and a light chain variable region with an amino acid sequence as described in SEQ ID NO:30; 4) comprising a heavy chain variable region with an amino acid sequence as described in SEQ ID NO:37 and a light chain variable region with an amino acid sequence as described in SEQ ID NO:38; 5) comprising a heavy chain variable region with an amino acid sequence as described in SEQ ID NO:45 and a light chain variable region with an amino acid sequence as described in SEQ ID NO:46.

[0024] Those skilled in the art will readily recognize that, based on the amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody specifically disclosed in this invention, one or more amino acids can be added, deleted, or replaced using conventional protein engineering methods to obtain a conserved variant or fragment thereof, while still maintaining specific binding to ASFV CD2V.

[0025] The present invention also provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0026] Due to the degeneracy of codons, based on the amino acid sequence of the monoclonal antibody described above, there can be a variety of nucleotide sequences that can encode the specific antigenic epitopes described in this invention.

[0027] The antibody nucleic acid molecules involved in this invention can be obtained using genetic engineering recombination technology or chemical synthesis methods. Those skilled in the art will readily understand that the variant sequences of the heavy chain variable region nucleotide sequence and / or light chain variable region nucleotide sequence obtained by mutation of the above-mentioned nucleic acid molecules provided in this invention through the addition, deletion, substitution, modification, etc., retain the ability to specifically bind to ASFV CD2V in single-chain antibodies, chimeric monoclonal antibodies, modified monoclonal antibodies, or other forms of monoclonal antibodies or antibody fragments encoded by these variant sequences. The beneficial effects achieved by this invention are:

[0028] This invention provides an African swine fever virus (ASFV) CD2V epitope peptide, the amino acid sequence of which is shown in any one of SEQ ID NO: 1-5. Experiments have demonstrated that the ASFV CD2V epitope peptide of this invention is reactive to ASFV-positive swine serum and specific anti-ASFV CD2V monoclonal antibodies, but unresponsive to ASFV-negative swine serum, exhibiting strong immunoreactivity and immunogenicity. This provides a new antigenic target for ASFV vaccine design, antibody development, and antibody detection kit development. The epitope peptide of this invention can be used as an immunogen to stimulate animals to produce anti-ASFV serum or anti-ASFV monoclonal antibodies. This invention provides five anti-ASFV CD2V monoclonal antibodies that can recognize the above-mentioned epitope peptides. The epitope peptide of this invention can be used to prepare ASFV vaccines and to prepare ASFV immunoassay reagents or kits, laying the foundation for further establishing efficient methods for detecting ASFV virus. Attached Figure Description

[0029] Figure 1 The expression of CD2V recombinant protein in the Bac-to-Bac baculovirus expression system; where A. SDSPAGE results of protein expression; B. Western blotting results of protein expression; C. IFA analysis results;

[0030] Figure 2 Purification of CD2V recombinant protein; including: A. SDSPAGE results of protein expression; B. Western blotting results of protein expression; C. Dot blotting analysis results; D. CD2V recombinant protein under electron microscopy, forming virus-like particles;

[0031] Figure 3Animal immunization with CD2V recombinant protein;

[0032] Figure 4 The predicted transmembrane region of the CD2V protein;

[0033] Figure 5 The results of Bepipred software prediction for CD2V protein;

[0034] Figure 6 The results of Lasergene secondary structure prediction for the CD2V protein;

[0035] Figure 7 It is an ASFV CD2V overlapping polypeptide;

[0036] Figure 8 The purpose of this study was to preliminarily identify CD2V protein B-cell epitopes using a scanning peptide assay. Specifically, AE and FJ were used to identify the reaction of 18 short peptides with CD2V monoclonal antibodies using i-ELISA (AE) and dot-ELISA (FJ), respectively.

[0037] Figure 9 To validate the truncation of the CD2V protein B-cell epitope; specifically, A. secondary structure analysis of peptide No. 14 was performed using DNAstar software; B. secondary structure analysis of peptide No. 18 was performed using DNAstar software; C. the reaction of the monoclonal antibody with short peptides No. 14 and No. 18 was detected by i-ELISA; E. (C) the reaction of the monoclonal antibody with short peptides No. 14 and No. 18 was detected by spot ELISA. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents involved are all commercially available conventional reagents; unless otherwise specified, the test methods involved are all conventional methods.

[0039] Example 1: Construction of the ASFV CD2V prokaryotic expression vector

[0040] The EP402R gene of the African swine fever virus isolate from Anhui, China (GenBank: MK128995.1) encodes the CD2V protein. A recombinant CD2V protein, namely CD2V-dimer, was constructed by tandemly linking two amino acid sequences of ASFV EP402R using a flexible linker (GGGGSGGGGSGGGGS). The nucleotide sequence of CD2V-dimer was optimized for codon bias using a eukaryotic baculovirus expression system and synthesized (Shanghai Sangon Biotech Co., Ltd.). The optimized sequence is shown in SEQ ID NO:6. The synthesized target gene is stored in the pFastBacI vector.

[0041] Example 2: Secretory expression of ASFV CD2V in a baculovirus expression system

[0042] Transform the recombinant positive pFastBac plasmid into DH10Bac TM E. coli competent cells, DH10Bac TM The transformation method for E. coli competent cells is the same as that for general TOP10 E. coli competent cells, except that after adding antibiotic-free medium, the cells should be cultured at 37°C with shaking at 200-225 rpm for 4-5 hours. After shaking culture, the bacterial suspension is plated with triple antibiotics (Kan (50 μg / ml), Tet (10 μg / ml), and Gen (7 μg / ml). Before plating, a mixture of 40 μl IPTG (0.6 M) and 100 μl X-gal (20 mg / ml) should be evenly spread on the triple antibiotic plate. Only 50-70 μl of bacterial suspension is needed for plating. After incubating the plates at 37°C for 48 hours, large, round white single colonies were picked and transferred to 10 ml of Kan, Tet, and Gen triple antibody LB medium. The plates were then incubated at 37°C with shaking at 200-220 rpm for 14-16 hours. The baculovirus plasmid rBacmid-gp67-ASFV CD2V was then extracted.

[0043] Healthy Sf21 cells were seeded in six-well plates at a density of approximately 2 × 10⁻⁶ cells / well. 6 / ml, secretory recombinant shuttle baculovirus rBacmid-gp67-ASFV CD2V was transfected into Sf21 insect cells, and cultured statically at 28℃. After 3 days, the supernatant was collected by centrifugation and labeled as P1 generation recombinant baculovirus. An untransfected empty cell control was also included. The P1 generation recombinant baculovirus solution was passaged at a volume fraction of 1% into 100×20mm cell culture dishes to obtain P2 and P3 generation recombinant baculoviruses. The supernatant of the P3 generation recombinant baculovirus was inoculated into 250ml Erlenmeyer flasks of Sf21 cells at a volume fraction of 1%-5%, with an uninoculated blank control. Cells were cultured at 28℃ and 120rpm for 3-5 days. The cell culture was centrifuged at 4℃ and 4000rpm for 30min, and the supernatant ASFVCD2V recombinant protein was collected. SDS-PAGE( Figure 1 A), Western blot ( Figure 1 B) and IFA Figure 1 C) The results are shown in the figure. Recombinant CD2V protein was well expressed at approximately 45 kDa in the baculovirus expression system.

[0044] Example 3: Purification of Recombinant ASFV CD2V Protein

[0045] The ASFV CD2V expression supernatant from Example 2 was purified using nickel affinity chromatography. The nickel affinity chromatography column was first equilibrated with 0.01M PBS buffer, then the sample was loaded. The ASFV CD2V supernatant was repeatedly flow-through the nickel affinity chromatography column at least three times. Impurities were washed with 0.01M PBS buffer containing 10mM and 50mM imidazole, and the target protein CD2V was eluted with 0.01M PBS buffer containing 100mM imidazole. The results are as follows. Figure 2 As shown. The CD2V target protein solution containing 100 mM imidazole was dialyzed to obtain the recombinant ASFV CD2V recombinant protein of the baculovirus expression system. Figure 2 AC). Electron microscopy (Figure 2D) results show that the ASFV CD2V recombinant protein is a virus-like particle with a size of approximately 10 nm.

[0046] Example 4: Animal immunization with ASFV CD2V

[0047] Recombinant ASFV CD2V protein was added to Freund's complete adjuvant (for the first immunization) and / or Freund's incomplete adjuvant (for the second, third, and fourth immunizations) and emulsified to prepare an immunoantigen. Three 6-8 week old female BALB / c mice were immunized via subcutaneous injection at multiple sites on the back, with an immunization dose of 5 μg per mouse; the immunization interval was 2 weeks. Fourteen days after the fourth immunization, polyclonal antibody serum was obtained by collecting blood from the tail vein of the mice.

[0048] The steps for detecting polyclonal antibody serum titer using ELISA are as follows: 1) Dissolve CD2VD in coating buffer at an appropriate concentration; 2) Add 100 μl of antigen to the corresponding well and incubate at room temperature for 2 h or at 4°C overnight; 3) Empty the liquid and pat dry any remaining liquid, then wash twice with 300 μl of washing buffer. 4) Add 300 μl of blocking buffer to each well and incubate for 1 h; 5) Empty the liquid and pat dry any remaining liquid, then wash twice with 300 μl of washing buffer; 6) Add 100 μl of primary antibody to each well and incubate at 37 °C for 1 h or at room temperature for 3 h; 7) Empty the liquid and pat dry any remaining liquid, fill each well with washing buffer, empty the liquid and pat dry, repeat 3 times; 8) Add 100 μl of secondary antibody to each well and incubate at room temperature for 1 h; 9) Empty the liquid and pat dry any remaining liquid, fill each well with washing buffer, empty the liquid and pat dry, repeat 3 times; 10) Soak in washing buffer for 5 min and pat dry any remaining liquid. This washing step is important for reducing background signal; 11) Fill each well with washing buffer, empty the liquid and pat dry, repeat 5 times; 12) Add 100 μl of substrate to each well, develop for 30 min and immediately take readings at 405-410 nm.

[0049] The results are as follows Figure 3 As shown, the titers in all three mice reached 1:6400 or higher, indicating that the recombinant CD2V protein has good immunogenicity.

[0050] Example 5: Preparation of Monoclonal Antibodies

[0051] Collect 2–5 × 10⁻⁵ well-growing sp2 / 0 tumor cells. 7 The cells were placed in 50ml centrifuge bottles. Mice were immunized with recombinant CD2V protein expressed by a baculovirus eukaryotic expression system. The immunogen was 20 μg of protein per mouse, with immunization intervals of 14 days. For the first immunization, the immunogen was fully emulsified with Freund's complete adjuvant, and for the subsequent three immunizations, the immunogen was fully emulsified with Freund's incomplete adjuvant. The immunization volume was 200 μL per mouse. Balb / c mice were exsanguinated 3-5 days after hyperimmunization, and serum was collected as a positive control. After cervical dislocation, the mice were disinfected with 75% alcohol for 5 minutes. Mouse spleen cells were fused with sp2 / 0 hybridoma cells using the PEG method. The suspended cells were dispersed into 10 96-well cell culture plates, with 220-250 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 approximately 9-12 days. Five monoclonal cell lines that reacted well to CD2V protein were screened using an indirect ELISA method (see Example 4), and named 22B3, 13G11, 7E12, 18A3, and 43C2, respectively. The variable region sequences of the five monoclonal antibodies were obtained using molecular cloning techniques and sequenced. The sequencing results are as follows:

[0052] The amino acid sequences of the heavy chain variable region and the light chain variable region of 22B3 are shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively. Further analysis revealed the amino acid sequences of the heavy chain variable regions VHCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 7-9; and the amino acid sequences of the light chain variable regions VLCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 10-12. The heavy chain and light chain variable regions of 22B3 are linked together by a linker (GGGGSGGGGSGGGGS). The nucleotide sequence encoding the heavy chain variable region of 22B3E3 is shown in SEQ ID NO. 47. The nucleotide sequence encoding the light chain variable region of 22B3E3 is shown in SEQ ID NO. 48.

[0053] The amino acid sequences of the heavy chain variable region and the light chain variable region of 13G11 are shown in SEQ ID NO. 21 and SEQ ID NO. 22, respectively. Further analysis revealed the amino acid sequences of the heavy chain variable regions VHCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 15-17; and the amino acid sequences of the light chain variable regions VLCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 18-20. The heavy chain and light chain variable regions of 13G11 are linked together by a linker (GGGGSGGGGSGGGGS). The nucleotide sequence encoding the heavy chain variable region of 13G11 is shown in SEQ ID NO. 49. The nucleotide sequence encoding the light chain variable region of 13G11 is shown in SEQ ID NO. 50.

[0054] The amino acid sequences of the heavy chain variable region and the light chain variable region of 7E12 are shown in SEQ ID NO. 29 and SEQ ID NO. 30, respectively. Further analysis revealed the amino acid sequences of the heavy chain variable region VHCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 23-25; and the amino acid sequences of the light chain variable region VLCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 26-28. The heavy chain variable region and the light chain variable region of 7E12 are linked together by a linker (GGGGSGGGGSGGGGS). The nucleotide sequence encoding the heavy chain variable region of 7E12 is shown in SEQ ID NO. 51. The nucleotide sequence encoding the light chain variable region of 7E12 is shown in SEQ ID NO. 52.

[0055] The amino acid sequences of the heavy chain variable region and the light chain variable region of 18A3 are shown in SEQ ID NO. 37 and SEQ ID NO. 38, respectively. Further analysis revealed the amino acid sequences of the heavy chain variable regions VHCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 31-33, and the amino acid sequences of the light chain variable regions VLCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 34-36, respectively. The heavy chain variable regions and the light chain variable regions of 18A3 are linked together using a linker (GGGGSGGGGSGGGGS). The nucleotide sequence encoding the 18A3 heavy chain variable region is shown in SEQ ID NO. 53. The nucleotide sequence encoding the 18A3 light chain variable region is shown in SEQ ID NO. 54.

[0056] The amino acid sequences of the heavy chain variable region and the light chain variable region of 43C2 are shown in SEQ ID NO. 45 and SEQ ID NO. 46, respectively. Further analysis revealed the amino acid sequences of the heavy chain variable regions VHCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 39-41, and the amino acid sequences of the light chain variable regions VLCDR1-3 of the monoclonal antibody as shown in SEQ ID NO. 42-44, respectively. The heavy chain and light chain variable regions of 43C2 are linked together by a linker (GGGGSGGGGSGGGGS). The nucleotide sequence encoding the heavy chain variable region of 43C2 is shown in SEQ ID NO. 55. The nucleotide sequence encoding the light chain variable region of 43C2 is shown in SEQ ID NO. 56.

[0057] Example 6 Bioinformatics Prediction of ASFV CD2V Linear B-cell Epitopes

[0058] The transmembrane region of CD2V was predicted using TMHMM Server v.2.0 software. The hydrophilicity and hydrophobicity of the CD2V protein were predicted using Bepipred software. The secondary structure, hydrophilicity, hydrophobicity, surface accessibility, and antigenicity of the ASFV CD2V protein were analyzed using the Protean module in Lasergene software.

[0059] Transmembrane region prediction results are as follows Figure 4 As shown, the extracellular region of CD2V is 1-206 aa, the transmembrane region is 207-229 aa, and the intracellular region is 230-360 aa.

[0060] Bepipred software prediction results are as follows: Figure 5As shown, the extracellular epitope peptides of the CD2V protein are: LDSNITNDNNDINGVSWNFFNNSF (12-35 aa), TCGKAGNFCECSNYSTSIYNI (40-60 aa), HNDVFDTTYQV (71-81 aa), NNGTN (116-120 aa), FVKYTNE (131-137 aa), NNSNINNF (145-152 aa), and LTLSSN (176-181 aa).

[0061] Lasergene secondary structure prediction results are as follows: Figure 6 As shown, the regions that readily form β-sheets and α-helices are 10-22 aa, 33-37 aa, 77-81 aa, 102-113 aa, 120-122 aa, 142-154 aa, 161-166 aa, 174-179 aa, and 199-209 aa; the regions with higher antigenic indices are 31-40 aa, 49-53 aa, 56-62 aa, 66-71 aa, 77-80 aa, 89-92 aa, 114-118 aa, 129-137 aa, 150-156 aa, 159-166 aa, 178-185 aa, and 195-198 aa. The regions with higher surface accessibility indices are 31-37aa, 130-137aa, 151-153aa, 159-164aa, 181-183aa, 195-198aa, and 206-209aa. Based on these three indicators, sequences 31-37aa, 77-80aa, 120-118aa, 150-154aa, 161-166aa, and 205-209aa are likely important B-cell antigenic determinants of the CD2V protein.

[0062] The predicted surface accessibility indices for sequences 77-80aa, 120-118aa, and 161-166aa are low, indicating that the probability of these amino acids being located on the CD2V surface is very low, and they are most likely embedded inside the CD2V protein. However, the N-terminal region, sequence 178-185aa, readily forms β-sheets and α-helical regions and exhibits a high antigenic index, high hydrophilicity, and high surface accessibility, suggesting that this region may be located on the surface of the CD2V protein.

[0063] Example 7 Design of ASFV CD2V overlapping peptides

[0064] Using the peptide scanning method, also known as the overlapping peptide method, the extracellular region of the ASFV CD2V protein was truncated into 18 short peptides. Peptides 1-17 are each 15 aa, and peptide 18 is 20 aa. Each short peptide has a 5 aa overlap. Figure 7The short peptide sequence was synthesized by Gill Biotech.

[0065] Table 1. ASFV CD2V overlapping polypeptide sequences

[0066] Name Sequence Name Sequence Name Sequence 1 DYWVSFNKTIILDSN 7 TNNCSLTIFPHNDVF 13 TNIYLNINDTFVKYT 2 ILDSNITNDNNDING 8 HNDVFDTTYQVVWNQ 14 FVKYTNESILEYNWN 3 NDINGVSWNFFNNSF 9 VVWNQIINYTIKLLT 15 EYNWNNSNINNFTAT 4 FNNSFNTLATCGKAG 10 IKLLTPATPPNITYN 16 NFTATCIINNTISTS 5 GKAGNFCECSNYST 11 NITYNCTNFLITCKK 17 TISTSNETTLINCTY 6 SNYSTSIYNITNNCS 12 ITCKKNNGTNTNIYL 18 INCTYLTLSSNYFYTFFKLY

[0067] Example 8 Identification of ASFV CD2V B cell epitopes

[0068] Add 100 μl of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) to 1 mg of 18 overlapping peptides, tighten the centrifuge tube cap, vortex and mix for 10 seconds to prepare a 1 mg / μL peptide stock solution. The short peptide stock solution was diluted to 2.5 μg / ml with CBS buffer (pH 9.6) and coated onto a 96-well microplate. Incubation was performed overnight at 4°C. The next day, the plates were washed three times with PBST buffer. Each well was blocked with 300 μl of 5% skim milk solution and incubated at 37°C for 2 h. Five mouse monoclonal antibodies against CD2V protein, diluted 1:2000 with 5% skim milk, were added as positive and negative controls. 50 μl of ASFV-positive and ASFV-negative swine serum were added to each well and incubated at 37°C for 1 h. The plates were washed three times with PBST buffer, and HRP-labeled goat anti-mouse or HRP-labeled goat anti-swine secondary antibody was added. Incubation was performed at 37°C for 1 h. The plates were washed five times with PBST buffer, and substrate chromogenic solution was added. The chromogenic development time was no more than 10 min, and then the chromogenic development was stopped with 2M sulfuric acid. Finally, the absorbance at 450 nm was read using a microplate reader.

[0069] like Figure 8 As shown, through three repeated experiments, the reaction of the anti-CD2V murine monoclonal antibody with the 18 short peptides was as follows: short peptide 14 showed a strong reaction with 22B3, 13G11, 7E12, 18A3, and 43C2, with OD values ​​of [missing information]. 450 Greater than 2, 18 reacts strongly with 22B3, 7E12, 18A3 and 43C2, its OD 450 Around 1, No. 18 and No. 13G11 showed a weaker reaction, with their OD... 450 Only around 0.5 ( Figure 8 In the AE (Acid Assay), 19 is the positive control and 20 is the negative control. Among them, the amino acid sequence of peptide 14 is FVKYTNESILEYNWN (147-161aa), and the amino acid sequence of peptide 18 is INCTYLTLSSNYFYTFFKLY (187-206aa).

[0070] The above experimental results show that the ASFV CD2V epitope peptide of the present invention is responsive to ASFV-positive porcine serum and specific anti-ASFV CD2V monoclonal antibodies, but not responsive to ASFV-negative porcine serum, exhibiting strong immunoreactivity and immunogenicity. This provides a new antigen target for ASFV vaccine design, antibody development, and antibody detection kit development.

[0071] Due to the degeneracy of codons, based on the amino acid sequence of the epitope peptides described above, there can be multiple nucleotide sequences capable of encoding the specific antigenic epitopes described in this invention. The epitope peptides, nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria of this invention are all beneficial for the prevention and / or treatment of African swine fever (ASF). Therefore, the epitope peptides, nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria of this invention can be used to prepare drugs for the prevention and / or treatment of African swine fever virus. Preferably, the drug is a vaccine. The vaccine includes one or more of the epitope peptides shown in SEQ ID NO:1-5. Preferably, the drug is anti-ASFV serum or anti-ASFV monoclonal antibody. The anti-ASFV serum or anti-ASFV monoclonal antibody is prepared using one or more of the epitope peptides shown in SEQ ID NO:1-5 as an immunogen.

[0072] The epitope peptides, nucleic acid molecules, expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria of the present invention can be used to prepare immunoassay reagents or kits for African swine fever (ASFV). Those skilled in the art will understand that the kits provided by the present invention can immobilize epitope peptides on ELISA plates for the detection of ASFV-specific antibodies (such as serum antibodies), or can use one or more epitope peptides shown in SEQ ID NO:1-5 as immunogens to prepare anti-ASFV antibodies, which are then coated onto ELISA plates for the detection of ASFV CD2V protein.

[0073] Example 9: ASFV CD2V B cell epitope truncation

[0074] The two epitope sequences obtained in Example 8 were truncated, as shown in Table 1. Following the method of overlapping peptides, they were truncated into six peptides, which were then coupled to BSA. The specific coupling method is as follows: First, the BSA carrier protein was coupled to sulfo-SMCC (taking the coupling of 20 mg of peptide as an example; all reagent volumes were scaled proportionally according to the actual coupling amount in the experiment). 20 mg of BSA (Thermofisher, Cat No.: 30063572) was weighed and dissolved in 2 ml of ultrapure water to prepare a 10 mg / ml BSA solution. 10 mg of Sulfo-SMCC (Thermofisher, Cat No.: 22322) was weighed and dissolved in 2 ml of ultrapure water to prepare a 5 mg / ml Sulfo-SMCC solution. The two solutions were mixed in equal volumes and reacted at room temperature (25°C) for 60 min or at 37°C for 30 min. The reaction was carried out with a magnetic stirrer at a slow, uniform speed to avoid generating bubbles. After the reaction, the reaction solution is placed in a 10kD dialysis bag and dialyzed in PBS (pH 7.4) to remove excess Sulfo-SMCC. The solution is changed every 2 hours, at least 3-4 times, to ensure complete dialysis, thus obtaining the activated BSA carrier. Then, the peptide is coupled. 20mg of peptide is weighed and dissolved in 5ml of cross-linking buffer (0.1M PB, 0.15M NaCl) to prepare a 4mg / ml peptide solution (for poorly soluble peptides, ≤30% DMSO can be used). Generally, only 5-10mg of peptide is coupled, proportionally reducing the volume. The activated and dialyzed BSA carrier is mixed with the 4mg / ml peptide solution and reacted at room temperature (25℃) for 4 hours. Finally, the uncoupled peptide is dialyzed in PBS (pH 7.4) using a 10kD dialysis bag, changing the solution every 2 hours, at least 4 times. The mixture is then stirred and dialyzed to the appropriate concentration, aliquoted into small tubes, and stored at -20℃.

[0075] Table 1. Truncated sequences of CD2V B cell epitopes

[0076] serial number sequence serial number sequence 14-1 <![CDATA[ 147 FVKYT 151 ]]> 18-1 <![CDATA[ 187 INCTYLTL 194 ]]> 14-2 <![CDATA[ 151 GENERATION 156 ]]> 18-2 <![CDATA[ 195 SSNY 198 ]]> 14-3 <![CDATA[ 157 EYNWN 161 ]]> 18-3 <![CDATA[ 199 FYTFFKLY 206 ]]>

[0077] The conjugated peptide was used for indirect ELISA to identify the reaction between the truncated peptide and the monoclonal antibody. For example... Figure 9 As shown in C and 9E, short peptides 14-1 and 14-3 can react with monoclonal antibodies 18A3 and 7E12. Compared to the secondary structure of peptide 14 ( Figure 9 A) 14-1 and 14-3 possess β-sheet and turn structures, belonging to the hydrophilic region, exhibiting high antigenicity and surface accessibility greater than zero, indicating that these two peptides are located extracellularly. While 14-2 also possesses high antigenicity, its surface accessibility is less than zero, suggesting that the 14-2 region is folded inward and thus cannot be recognized. 18-1 and 18-3 do not react with any monoclonal antibodies. Figure 9 D and 9F), while 18-2 reacted with 18A3 and 7E12 monoclonal antibodies, a result consistent with that obtained from secondary structure analysis. Figure 9 B). Therefore, the B-cell epitope of CD2V protein is 14-1 (B). 147 FVKYT 151 ),14-3( 157 EYNWN 161 ) and 18-2 ( 195 SSNY 198 ). <110> Zhengzhou University, Henan Zhongze Biotechnology Co., Ltd. <120> African swine fever virus CD2V epitope peptide and its application, anti-African swine fever virus CD2V monoclonal antibody <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <221> Antigenic peptide (14-1) <400> 1 Phe Val Lys Tyr Thr 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <221> Antigenic peptide (14-3) <400> 2 Glu Tyr Asn Trp Asn 1 5 <210> 3 <211> 4 <212> PRT <213> Artificial sequence <221> Antigenic peptide (18-2) <400> 3 Ser Ser Asn Tyr 1 <210> 4 <211> 15 <212> PRT <213> Artificial sequence <221> Antigen peptide (No. 14) <400> 4 Phe Val Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn Trp Asn 1 5 10 15 <210> 5 <211> 20 <212> PRT <213> Artificial sequence <221> Antigen peptide (No. 18) <400> 5 Ile Asn Cys Thr Tyr Leu Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe 1 5 10 15 Phe Lys Leu Tyr 20 <210> 6 <211> 1350 <212> DNA <213> Artificial sequence <221> ASFV EP402R <400> 6 ggatccatga agttcctggt taacgtggct ctggtgttca tggtggtgta catctcctac 60 atctacgcca tgatcatcct catcttcctg atcttcagca acatcgtcct gtccatcgat 120 tactgggtca gcttcaacaa gacaatcatc ttggacagca acatcactaa cgacaacaac 180 gacatcaatg gtgtgtcctg gaacttcttc aacaacagtt tcaacaccct tgctacctgc 240 ggcaaggctg ggaacttctg tgaatgcagc aactactcta ccagcatcta caacatcacc 300 aacaactgca gcctgaccat cttcccccac aacgacgtgt tcgacaccac ataccaggtg 360 gtgtggaacc agatcatcaa ctatacaatc aagctgctga cccctgccac cccacctaac 420 atcacctaca actgtaccaa cttcctcatc acctgcaaga agaacaacgg cacaaacacg 480 aacatctacc tgaacatcaa cgacaccttc gtgaagtaca ccaacgagtc catcctggag 540 tacaactgga acaactccaa catcaacaac ttcaccgcca cctgcatcat caacaacaca 600 atcagcacta gcaacgaaac caccctgatc aactgtacct acctgaccct gtcttctaac 660 tacttctaca ccttcttcaa gctgtacggc ggaggtggtt ccggtggtgg cggcagcggc 720 ggtggtggct ccatgatcat cctgatcttc ctcatcttct ccaacatcgt gctgagcatc 780 gactactggg tgagcttcaa caagaccatc atcctcgact ccaacatcac taacgacaac 840 aacgacatca acggcgtttc ctggaacttc ttcaacaact ccttcaacac tctcgccacc 900 tgtggcaagg ccggcaactt ctgtgagtgt agcaactact cgaccagcat ctacaacatt 960 accaacaact gctccctcac catcttcccc cacaacgacg tgttcgacac aacttaccag 1020 gtggtctgga accagatcat cactacacc atcagctgc tcacccagc caccctcct 1080 aacatcacct acaactac aaacttcctg atcacatgta agagaacaa cggcaccac 1140 accacatct acctgaacat caacgacaca tcgtgaagt acaccacga gtccatcctg 1200 gagtacaact ggacaacag cacacaac aacttcacag ctacctgcat catcacaac 1260 accatcagca ccagcaacga gaccaccctc atcaactgca cttacctgac cctgagctcc 1320 aactactct acacctctt caagctgtac 1350 <210> 7 <211> 13 <212> PRT <221> VHCDR1(22B3) <400> 7 Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 10 <210> 8 <211> 16 <212> PRT <221> VHCDR2(22B3) <400> 8 Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys Gly 1 5 10 15 <210> 9 <211> 10 <212> PRT <221> VHCDR3(22B3) <400> 9 Arg Tyr Arg Tyr Asp Ala Trp Phe Ala Tyr 1 5 10 <210> 10 <211> 10 <212> PRT <221> VLCDR1 (22B3) <400> 10 Arg Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 11 <211> 7 <212> PRT <221> VLCDR2 (22B3) <400> 11 Ala Thr Ser Asn Leu Ala Ser 1 5 <210> 12 <211> 7 <212> PRT <221> VLCDR3 (22B3) <400> 12 Gln Gln Trp Ser Ser Asn Pro 1 5 <210> 13 <211> 120 <212> PRT <221> Heavy chain variable region (22B3) <400> 13 Met His Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly 1 5 10 15 Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 20 25 30 Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp 35 40 45 Val Ala Ser Ile Ser Ser Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Phe Cys 85 90 95 Ala Arg Arg Tyr Arg Tyr Asp Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Lys 115 120 <210> 14 <211> 105 <212> PRT <221> light chain variable region (22B3) <400> 14 Met Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val 1 5 10 15 Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr 20 25 30 Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Thr Ser 35 40 45 Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 50 55 60 Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Val Ala 65 70 75 80 Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Arg Gly Arg Ser Val 85 90 95 Glu Ala Pro Ser Trp Lys Ser Asn Gly 100 105 <210> 15 <211> 14 <212> PRT <221> VHCDR1(13G11) <400> 15 Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly Tyr Ser Trp His 1 5 10 <210> 16 <211> 14 <212> PRT <221> VHCDR2(13G11) <400> 16 Tyr Ile His Tyr Ser Gly Val Thr Asn Tyr Asn Pro Ser Leu 1 5 10 <210> 17 <211> 13 <212> PRT <221> VHCDR3(13G11) <400> 17 Ala Pro Leu Tyr Tyr Gly Asn Tyr Val Trp Phe Ser Tyr 1 5 10 <210> 18 <211> 11 <212> PRT <221> VLCDR1(13G11) <400> 18 Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala 1 5 10 <210> 19 <211> 7 <212> PRT <221> VLCDR2 (13G11) <400> 19 Asn Ala Lys Thr Leu Ala Asp 1 5 <210> 20 <211> 9 <212> PRT <221> VLCDR3 (13G11) <400> 20 Gln His Phe Trp Ser Thr Pro Trp Thr 1 5 <210> 21 <211> 125 <212> PRT <221> Heavy chain variable region (13G11) <400> 21 Met Ile Met Val Gln Ser Gln Glu Ser Gly Pro Asp Leu Val Lys Pro 1 5 10 15 Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr 20 25 30 Ser Gly Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu 35 40 45 Glu Trp Met Gly Tyr Ile His Tyr Ser Gly Val Thr Asn Tyr Asn Pro 50 55 60 Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 65 70 75 80 Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg Ala Pro Leu Tyr Tyr Gly Asn Tyr Val Trp Phe Ser 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Lys 115 120 125 <210> 22 <211> 113 <212> PRT <221> Light chain variable region (13G11) <400> 22 Met Thr Cys Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala 1 5 10 15 Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile 20 25 30 His Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln 35 40 45 Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Ser Gln Tyr Ser Leu Lys Ile Asn Ser 65 70 75 80 Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser 85 90 95 Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Asn Gln Thr Asp 100 105 110 Lys <210> 23 <211> 13 <212> PRT <221> VHCDR1(7E12) <400> 23 Lys Thr Ser Gly Tyr Ile Phe Thr Ser Tyr Trp Ile His 1 5 10 <210> 24 <211> 17 <212> PRT <221> VHCDR2(7E12) <400> 24 Arg Ile Tyr Pro Gly Thr Gly Ser Thr Tyr Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 25 <211> 11 <212> PRT <221> VHCDR3(7E12) <400> 25 Gly Lys Tyr Gly Asn Leu Tyr Tyr Phe Asp Tyr 1 5 10 <210> 26 <211> 15 <212> PRT <221> VLCDR1(7E12) <400> 26 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 27 <211> 7 <212> PRT <221> VLCDR2 (7E12) <400> 27 Leu Val Ser Asn Leu Glu Ser 1 5 <210> 28 <211> 9 <212> PRT <221> VLCDR3 (7E12) <400> 28 Gln His Ile Arg Glu Leu Thr Arg Ser 1 5 <210> 29 <211> 122 <212> PRT <221> Heavy chain variable region (7E12) <400> 29 Met Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 1 5 10 15 Ala Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ile Phe Thr Ser 20 25 30 Tyr Trp Ile His Trp Val Lys Gln Arg Ser Gly Gln Gly Leu Glu Trp 35 40 45 Ile Ala Arg Ile Tyr Pro Gly Thr Gly Ser Thr Tyr Tyr Asn Glu Lys 50 55 60 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Gln Leu Ser Ser Leu Lys Ser Glu Asp Ser Ala Val Tyr Phe 85 90 95 Cys Ala Arg Gly Lys Tyr Gly Asn Leu Tyr Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Lys 115 120 <210> 30 <211> 112 <212> PRT <221> light chain variable region (7E12) <400> 30 Met Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu 1 5 10 15 Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr 20 25 30 Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro 50 55 60 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile 65 70 75 80 His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile 85 90 95 Arg Glu Leu Thr Arg Ser Glu Gly Ala Pro Ser Trp Lys Ser Asn Gly 100 105 110 <210> 31 <211> 13 <212> PRT <221> VHCDR1(18A3) <400> 31 Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Tyr Ile His 1 5 10 <210> 32 <211> 17 <212> PRT <221> VHCDR2(18A3) <400> 32 Trp Ile Phe Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 33 <211> 7 <212> PRT <221> VHCDR3(18A3) <400> 33 Thr Gly Arg Val Phe Ala Tyr 1 5 <210> 34 <211> 11 <212> PRT <221> VLCDR1(18A3) <400> 34 Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser 1 5 10 <210> 35 <211> 7 <212> PRT <221> VLCDR2(18A3) <400> 35 Ala Ala Ser Thr Leu Asp Ser 1 5 <210> 36 <211> 9 <212> PRT <221> VLCDR3 (18A3) <400> 36 Leu Gln Tyr Ala Ser Tyr Pro Trp Thr 1 5 <210> 37 <211> 107 <212> PRT <221> Heavy chain variable region (18A3) <400> 37 Met Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly 1 5 10 15 Tyr Ser Phe Thr Ser Tyr Tyr Ile His Trp Val Lys Gln Arg Pro Gly 20 25 30 Gln Gly Leu Glu Trp Ile Gly Trp Ile Phe Pro Gly Ser Gly Asn Thr 35 40 45 Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr 50 55 60 Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp 65 70 75 80 Ser Ala Val Tyr Phe Cys Ala Gln Thr Gly Arg Val Phe Ala Tyr Trp 85 90 95 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Lys 100 105 <210> 38 <211> 106 <212> PRT <221> Light chain variable region (18A3) <400> 38 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Glu Arg Val 1 5 10 15 Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser Trp 20 25 30 Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile Tyr Ala Ala 35 40 45 Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly Ser Arg Ser 50 55 60 Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser Glu Asp Phe 65 70 75 80 Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Trp Thr Phe Gly 85 90 95 Gly Gly Thr Lys Leu Glu Ser Asn Gly Glu 100 105 <210> 39 <211> 13 <212> PRT <221> VHCDR1(43C2) <400> 39 Lys Ala Thr Gly Tyr Thr Phe Ser Thr Tyr Trp Ile Glu 1 5 10 <210> 40 <211> 17 <212> PRT <221> VHCDR2(43C2) <400> 40 Glu Ile Leu Pro Gly Gly Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 41 <211> 15 <212> PRT <221> VHCDR3 (43C2) <400> 41 Val Arg Tyr Gly Asn Tyr Gly Gly Asn Tyr Tyr Ala Met Asp Tyr 1 5 10 15 <210> 42 <211> 15 <212> PRT <221> VLCDR1 (43C2) <400> 42 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr Met His 1 5 10 15 <210> 43 <211> 7 <212> PRT <221> VLCDR2 (43C2) <400> 43 Leu Val Ser Asn Leu Glu Ser 1 5 <210> 44 <211> 9 <212> PRT <221> VLCDR3 (43C2) <400> 44 Gln His Ile Arg Glu Leu Thr Arg Ser 1 5 <210> 45 <211> 123 <212> PRT <221> Heavy chain variable region (43C2) <400> 45 Met Ala Ala Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Thr Tyr Trp 20 25 30 Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly 35 40 45 Glu Ile Leu Pro Gly Gly Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys 50 55 60 Gly Lys Ala Met Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Met 65 70 75 80 Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Arg Tyr Gly Asn Tyr Gly Gly Asn Tyr Tyr Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 46 <211> 107 <212> PRT <221> light chain variable region (43C2) <400> 46 Met Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu 1 5 10 15 Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr 20 25 30 Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro 50 55 60 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile 65 70 75 80 His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile 85 90 95 Arg Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys 100 105 <210> 47 <211> 359 <212> DNA <221> Heavy chain variable region (22B3) <400> 47 ttcatgtgca gctgcaggag tcagggggag gcttagtgaa gcctggaggg tccctgaaac 60 tctcctgtgc agcctctgga ttcactttca gtagctatgc catgtcttgg gttcgccaga 120 ctccagagaa gaggctggag tgggtcgcat ccattagtag tggtggtagc acctactatc 180 cagacagtgt gaagggccga ttcaccatct ccagagataa tgccaggaac atcctgtacc 240 tgcaaatgag cagtctgagg tctgaggaca cggccatgta tttctgtgca agaagatata 300 ggtacgacgc ctggtttgct tactggggcc aagggaccac ggtcaccgtc tcctcaaaa 359 <210> 48 <211> 327 <212> DNA <221> Light chain variable region (22B3) <400> 48 gtttgacatt gagccaccca gtctccagca atcctgtctg catctccagg ggagaaggtc 60 acaatgactt gcagggccag ctcaagtgta agttacatgc actggtacca gcagaagcca 120 ggatcctccc ccaaaccctg gatttatgcc acatccaacc tggcttctgg agtccctgct 180 cgcttcagtg gcagtgggtc tgggacctct tactctctca caatcagcag agtggaggct 240 gaagatgttg ccacttatta ctgccagcag tggagtagta acccacgtgg acgttcggtg 300 gaggcaccaa gctggaaatc aaacgga 327 <210> 49 <211> 374 <212> DNA <221> Heavy chain variable region (13G11) <400> 49 tttttgagga gacggtgacc gtggtccctt ggccccagta agaaaaccag acgtagttac 60 catagtagag cggggccctt gcacagtaat atgtggctgt gtcctcagta gtcacagaat 120 tcaactgcag gaagaactga ttcttggatg tgtctcgagt gatggagatt cgacttttga 180 gagatgggtt gtagttagtg acaccactgt agtgtatgta gcccatccat tccagtttgt 240 ttcctggaaa ctgccggatc cagtgccagc tataaccact ggtgatggag tagccagtga 300 cagtgcaggt gagtgaaagt gactgagaag gtttcaccag gtcaggtcct gactcctgcg 360 actgcaccat gata 374 <210> 50 <211> 338 <212> PRT <221> Light chain variable region (13G11) <400> 50 tttatccgtt tgattttcca gcttggtgcc tccaccgaac gtccacggag tactccaaaa 60 atgttgacag taataactcc caaaatcttc aggctgcagg ctgttgatct tgagagaata 120 ttgtgatcct gatccactgc cactgaacct tgatggcaca ccatctgcta aggtttttgc 180 attatagacc aggagctgag gagattttcc ctgtttctgc tgataccatg ctaaataatt 240 gtgaatattc ccacttgctc gacatgtgat ggtgacagtt tctcccacag atgcagatag 300 ggaggctgga gactgggtga gctcaatgtc acatgtca 338 <210> 51 <211> 365 <212> DNA <221> Heavy chain variable region (7E12) <400> 51 ttttgaggag acggtgaccg tggtcccttg gccccagtag tcaaagtagt acaggttacc 60 atacttccct cttgcacaga aatagacagc agagtcctca gatttcaggc tgctgagctg 120 catgtaggca gtgctggagg atttgtctgc agtcagtgtg gccttgccct tgaacttctc 180 attgtagtaa gtactaccag ttccaggata aatccttgca atccactcaa ggccctgtcc 240 agacctctgt tttacccagt gaatccagta gctggtgaag atgtatccag aagtcttgca 300 ggacagcttc actgaagccc caggcctcac cagctcagct ccagactgct gcagctgcac 360 ctcaa 365 <210> 52 <211> 339 <212> DNA <221> Light chain variable region (7E12) <400> 52 tttatccgtt tgatttccag cttggtgccc cctccgaacg tgtaagctcc ctaatgtgct 60 gacagtaata ggttgcagca tcctcctcct ccacaggatg gatgttgagg gtgaagtctg 120 tcccagaccc actgccactg aacctggcag ggaccccaga ttctaggttg gatacaagat 180 agatgaggag tctgggtggc tgtcctggtt tctgttggtt ccagtgcata taactatagc 240 cagatgtact gacacttttg ctggccctgt atgagatggt ggccctctgc cccagagata 300 cagctaagga agcaggagac tgggtgagct caatgtcaa 339 <210> 53 <211> 354 <212> DNA <221> Heavy chain variable region (18A3) <400> 53 ttttgaggag acggtgaccg tggtcccttg gccccagtaa gcaaaaaccc tcccagtttg 60 tgcacagaaa tagactgcag agtcctcaga tgtcaggctg ctgagctgca tgtaggctgt 120 gctggaggat gtgtctgccg tcagtgtggc cttgcccttg aacttctcat tgtacttagt 180 attaccactt ccaggaaaaa tccatccaat ccactcaagt ccctgtccag gcctctgctt 240 cacccagtgt atatagtagc ttgtgaagct gtagccagaa gccttgcagg atatcttcac 300 tgaagcccca ggcttcacca gctcaggtcc agactgctga gactgcacct tgac 354 <210> 54 <211> 335 <212> DNA <221> Light chain variable region (18A3) <400> 54 ttctccgttt gattccagct tggtgcctcc accgaacgtc cacggataac tagcatattg 60 tagacagtaa tagtctgcaa aatcttcaga ctcaaggctg ctgatggtga gagaataatc 120 tgacccagac ctactgccac tgaacctttt tgggacacca gaatctaaag tggatgcggc 180 gtagatcagg cgtttaatag ttccatctgg tttctgctga agccagctta agtaaccact 240 aatttcctga cttgcccgac aagtgagact gactctttct cccagagagg cagataagga 300 ggatggagac tgggtggctc aatgtcacaa tgtca 335 <210> 55 <211> 376 <212> DNA <221> Heavy chain variable region (43C2) <400> 55 tttgaggaga cggtgaccgt ggtcccttgg ccccagtagt ccatagcata gtaatttccc 60 ccgtagttac catacctcac tcttgcacag taatagacgg cagagtcctc agatgccagg 120 ctgctgagtt gcatgtaggc tgtgttggag gatgtatctg cagtgaacat ggccttgccc 180 ttgaacttct cattgtagtt agtactacca cctccaggta aaatctctcc aatccactca 240 aggccatgtc caggcctctg ttttacccac tctatccagt aggtactgaa tgtgtagcca 300 gtagccttgc aggatatttt cactgaggcc ccaggcttca tcagctcagc tccagactgc 360 tgcgctgcac ctcaaa 376 <210> 56 <211> 339 <212> DNA <221> light chain variable region (43C2) <400> 56 tgtctccgtt ttatttccag cttggtcccc cctccgaacg tgtaagctcc ctaatgtgct 60 gacagtaata ggttgcagca tcctcctcct ccacaggatg gatgttgagg gtgaagtctg 120 tcccagaccc actgccactg aacctggcag ggaccccaga ttctaggttg gatacaagat 180 agatgaggag tctgggtggc tgtcctggtt tctgttggtt ccagtgcata taactatagc 240 cagatgtact gacacttttg ctggccctgt atgagatggt ggccctctgc cccagagata 300 cagctaagga agcaggagac tgggtgagct caatgtcaa 339

Claims

1. A monoclonal antibody against African swine fever virus CD2V, characterized in that, The monoclonal antibody comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO:31-33, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NO:34-36, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:37, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

38.

3. A nucleic acid molecule, characterized in that, Its encoding is the monoclonal antibody as described in claim 1 or 2.

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