Structural domain specific monoclonal antibody and application thereof

Abstract

The invention provides the antibody specifically bound with the African swine fever virus CD2v protein and the application thereof, the antibody is high in titer and stable in property, can be bound with ASFV infected cells, and provides an important tool for ASFV detection, CD2v antigen or fragment detection and CD2v protein structure analysis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a domain-specific monoclonal antibody and application thereof, in particular to an antibody specifically binding to the first domain of the extracellular segment of CD2v protein of African swine fever virus and application thereof. BACKGROUND

[0002] African swine fever (ASF) is an acute, severe, highly contagious disease of pigs caused by African swine fever virus (ASFV) infection of domestic and wild pigs, with clinical features of high fever, anorexia, skin cyanosis and internal organ hemorrhage, short course of disease, and mortality rate up to 100%. ASF is a statutory report animal disease of the World Organization for Animal Health (OIE) and a class A animal disease in China, and is highly valued by countries around the world. Since the disease was discovered nearly a hundred years ago, domestic and foreign scholars have done a lot of research on ASF, but the research found that: the inactivated vaccine has no obvious effect, the attenuated vaccine has certain protective effect but poor safety, and there is no effective vaccine for preventing ASF and specific drug for treating the disease in the world at present. The control of ASF can only rely on rapid diagnosis, culling of sick animals, and effective quarantine measures and strict health measures.

[0003] ASFV is the only member of the Asfarviridae family and Asfivirus genus, and the only known DNA arbovirus. ASFV is a large and complex icosahedral, double-layered membrane double-stranded linear DNA virus (170-194 kb), with a virus particle diameter of about 260 nm, containing 151-167 open reading frames that can encode 150-200 proteins. ASFV mainly infects mononuclear cells and alveolar macrophages, and completes virus invasion through endocytosis and macropinocytosis, and its DNA replication, virus assembly and release are all carried out in macrophages.

[0004] CD2v protein is encoded by ASFV EP402R gene, is a glycoprotein of the outer envelope of African swine fever virus, and is a type I transmembrane protein expressed in the late stage of ASFV. CD2v protein is divided into four parts of N-terminal signal peptide, extracellular region, transmembrane region and C-terminal intracellular domain, the extracellular region has two immunoglobulin-like domains (IG) and the amino acid sequence is similar to the host's CD2 protein, which can be expressed in T cells and NK cells; the intracellular region has no obvious similarity with the host's CD2 protein.

[0005] As a major outer membrane protein of ASFV, CD2v protein enables virus-infected cells and extracellular viral particles to adhere to erythrocytes, playing a crucial role in viral transmission and replication. Furthermore, the extracellular domain of CD2v is a key protein leading to blood cell adhesion. As one of the glycosylated proteins in African swine fever virus, it plays a significant role in the viral escape mechanism. Simultaneously, based on the HAI characteristics of CD2v, researchers have classified the virus into serotypes based on the existing ASFV strains classified as genotype I / II according to the P72 gene. To date, several ASFV gene-deleted vaccine candidate strains have been obtained internationally using genetic engineering techniques. Among them, attenuated strains with a CD2v gene deletion can provide protection for immunized pigs. Therefore, research based on the CD2v protein is of great significance. However, as a major membrane protein and important antigenic molecule of ASFV, the molecular structure of CD2v remains unresolved. Therefore, the preparation of its monoclonal antibodies, especially monoclonal antibodies with different domains, can provide important tools for ASFV detection, CD2v protein structural analysis, and vaccine development. Summary of the Invention

[0006] To address one of the aforementioned technical problems in the prior art, this invention provides a domain-specific monoclonal antibody and its application, particularly an antibody that specifically binds to the first extracellular domain of the CD2v protein of African swine fever virus and its application.

[0007] The first aspect of this invention provides an antibody or antigen-binding fragment thereof that specifically binds to the CD2v protein of African swine fever virus, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity-determining regions (CDRs): (a1) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO:36; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO:38; (a2) The heavy chain variable regions containing CDR-H1, CDR-H2 and CDR-H3, and / or the light chain variable regions containing CDR-L1, CDR-L2 and CDR-L3, wherein, compared with the heavy chain variable regions and / or light chain variable regions described in (a1), at least one CDR contains a mutation, said mutation being a substitution, deletion or addition of one or more amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids).

[0008] In some implementations, the substitution is a conservative substitution.

[0009] In some embodiments, the antibody or its antigen-binding fragment specifically binds to the extracellular domain SD1 of the African swine fever virus CD2v protein.

[0010] In some embodiments, the first domain SD1 has the amino acid sequence represented by amino acids 26 to 111 in SEQ ID NO:4 or a variant thereof, the variant having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or having one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; in some embodiments, the substitutions are conservative substitutions (e.g., substitutions, deletions, or additions of one, two, or three amino acids).

[0011] In some implementations, the complementary decision region is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0012] In some embodiments, the antibody or its antigen-binding fragment includes the following heavy chain variable regions and / or light chain variable regions, wherein the complementarity-determining regions are defined according to the IMGT numbering system: The heavy chain variable region contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:40 or a variant thereof; CDR-H2 with the sequence of SEQ ID NO:41 or a variant thereof; CDR-H3 with the sequence of SEQ ID NO:42 or a variant thereof; and / or, the light chain variable region contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:52 or a variant thereof; CDR-L2 with the sequence of SEQ ID NO:53 or a variant thereof; CDR-L3 with the sequence of SEQ ID NO:54 or a variant thereof; The variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; in some embodiments, the substitutions are conservative substitutions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0013] In some embodiments, the antibody or its antigen-binding fragment includes the following heavy chain variable regions and / or light chain variable regions, wherein the complementarity-determining regions are defined according to the Kabat numbering system: The heavy chain variable region contains the following three CDRs: CDR-H1 with sequence SEQ ID NO:43 or a variant thereof; CDR-H2 with sequence SEQ ID NO:44 or a variant thereof; CDR-H3 with sequence SEQ ID NO:45 or a variant thereof; and / or, the light chain variable region contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:55 or a variant thereof; CDR-L2 with sequence SEQ ID NO:56 or a variant thereof; CDR-L3 with sequence SEQ ID NO:57 or a variant thereof; The variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; in some embodiments, the substitutions are conservative substitutions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0014] In some embodiments, the antibody or its antigen-binding fragment includes the following heavy chain variable regions and / or light chain variable regions, wherein the complementarity-determining regions are defined according to the Chothia numbering system: The heavy chain variable region contains the following three CDRs: CDR-H1 with sequence SEQ ID NO:46 or a variant thereof; CDR-H2 with sequence SEQ ID NO:47 or a variant thereof; CDR-H3 with sequence SEQ ID NO:48 or a variant thereof; and / or, the light chain variable region contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:55 or a variant thereof; CDR-L2 with sequence SEQ ID NO:56 or a variant thereof; CDR-L3 with sequence SEQ ID NO:57 or a variant thereof; The variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; in some embodiments, the substitutions are conservative substitutions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0015] In some embodiments, the antibody or its antigen-binding fragment includes the following heavy chain variable regions and / or light chain variable regions, wherein the complementarity-determining regions are defined according to the Contact numbering system: The heavy chain variable region contains the following three CDRs: CDR-H1 with sequence SEQ ID NO:49 or a variant thereof; CDR-H2 with sequence SEQ ID NO:50 or a variant thereof; CDR-H3 with sequence SEQ ID NO:51 or a variant thereof; and / or, the light chain variable region contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:58 or a variant thereof; CDR-L2 with sequence SEQ ID NO:59 or a variant thereof; CDR-L3 with sequence SEQ ID NO:60 or a variant thereof; The variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; in some embodiments, the substitutions are conservative substitutions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises: The heavy chain variable region contains the sequence shown in SEQ ID NO:36 or a variant thereof and / or the light chain variable region contains the sequence shown in SEQ ID NO:38 or a variant thereof; The variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; in some embodiments, the substitutions are conservative substitutions.

[0017] In some embodiments, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a porcine antibody.

[0018] In some embodiments, the antibody is of the IgA, IgD, IgE, IgG, or IgM type.

[0019] In some embodiments, the antibody is of the IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 type.

[0020] In some embodiments, the antibody or its antigen-binding fragment further comprises a constant region derived from or derived from mouse immunoglobulins, or a constant region derived from or derived from porcine immunoglobulins.

[0021] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from or originating from mouse immunoglobulins.

[0022] In some embodiments, the antibody or its antigen-binding fragment contains a wild-type Fc region, or contains a mutated or chemically modified Fc region that has altered effector functions compared to the wild-type Fc region.

[0023] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from or originating from mouse IgG2a.

[0024] In some embodiments, the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from or originating from mouse immunoglobulins.

[0025] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region derived from or originating from mouse Igκ.

[0026] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO:61 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO:61.

[0027] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region or a variant thereof as shown in SEQ ID NO:63, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO:63.

[0028] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO:61 and a light chain constant region as shown in SEQ ID NO:63.

[0029] In some embodiments, the antibody or its antigen-binding fragment comprises: The heavy chain including the heavy chain variable region shown in SEQ ID NO:36 and the heavy chain constant region shown in SEQ ID NO:61, and the light chain including the light chain variable region shown in SEQ ID NO:38 and the light chain constant region shown in SEQ ID NO:63.

[0030] In some embodiments, the antibody or its antigen-binding fragment is ScFv, Fab, Fab', Fab'-SH, F(ab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, bispecific antibody, or multispecific antibody.

[0031] In some embodiments, the antibody or its antigen-binding fragment is labeled.

[0032] In some embodiments, the antibody or its antigen-binding fragment carries a detectable marker, which can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable marker described above can be linked to the antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus CD2v protein disclosed herein via linkers of different lengths to reduce potential steric hindrance.

[0033] A second aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof as described in the first aspect, its heavy chain and / or light chain, and its heavy chain variable region and / or light chain variable region. According to codon degeneracy in the art, in some embodiments, the nucleotide sequence can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0034] In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding the antibody heavy chain variable region and / or a nucleic acid molecule encoding the antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:37, (ii) a sequence substantially identical to SEQ ID NO:37 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:37, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence of (i) or (ii) above; and / or, the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence shown in SEQ ID NO:39, (v) a sequence substantially identical to SEQ ID NO:39 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to SEQ ID NO:39, or a sequence having one or more nucleotide substitutions), or (vi) a degenerate sequence of (iv) or (v) above.

[0035] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the second aspect.

[0036] In some embodiments, the vector is a cloning vector or an expression vector.

[0037] In some embodiments, the vector is an expression vector. In some embodiments, the expression vector may include eukaryotic expression vectors and / or prokaryotic expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0038] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0039] A fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect or the vector described in the third aspect.

[0040] In some embodiments, the host cell does not involve reproductive material.

[0041] In some embodiments, the host cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus CD2v protein as disclosed above, a chimeric antigen receptor or a multispecific antibody or antigen-binding fragment thereof. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0042] The fifth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first aspect, comprising culturing the host cell as described in the fourth aspect under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0043] The sixth aspect of the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect, or a nucleic acid molecule as described in the second aspect, or a carrier as described in the third aspect, or a host cell as described in the fourth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0044] A seventh aspect of the present invention provides a detection kit containing the antibody or antigen-binding fragment thereof described in the first aspect, or the nucleic acid molecule described in the second aspect, or the vector described in the third aspect, or the host cell described in the fourth aspect, the kit being used to detect the presence or level of ASFV or a protein containing the first extracellular domain SD1 of the CD2v protein of African swine fever virus in a sample.

[0045] The eighth aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, or the nucleic acid molecule described in the second aspect, or the carrier described in the third aspect, the host cell described in the fourth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a reagent, said reagent being used for one or more of the following purposes: 1) Detect the presence or level of African swine fever virus or protein containing the first extracellular domain SD1 of the African swine fever virus CD2v protein in the sample; 2) Prevention and / or treatment of diseases associated with African swine fever virus.

[0046] In some embodiments, the first domain SD1 has the amino acid sequence represented by amino acids 26 to 111 in SEQ ID NO:4 or a variant thereof, the variant having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or having one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; in some embodiments, the substitutions are conservative substitutions (e.g., substitutions, deletions, or additions of one, two, or three amino acids).

[0047] In some embodiments, the disease associated with the African swine fever virus is African swine fever.

[0048] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a monoclonal antibody that specifically binds to the first extracellular domain of the CD2v extracellular domain of African swine fever virus (ASFV). This antibody exhibits high titer and stability, can bind to ASFV-infected cells, and can be used for detection by ELISA, flow cytometry, Western blot, and immunohistochemistry. The variable region sequence of the monoclonal antibody provided by this invention can be used to construct complete genetically engineered antibodies and for the expression of Fab antibodies, providing an important tool for ASFV detection, detection of CD2v antigens or fragments, and structural analysis of the CD2v protein. Attached Figure Description

[0049] Figure 1 The SDS-PAGE quantification results of CD2v FL\SD1\SD2 recombinant proteins are presented.

[0050] Figure 2 The binding of CD2v FL\SD1\SD2 recombinant protein to African swine fever positive serum was demonstrated.

[0051] Figure 3 The results of ELISA detection of CD2v FL, SD1, and SD2 proteins were presented in the culture supernatant of hybridoma cells E1Y003.

[0052] Figure 4 The results of SDS-PAGE electrophoresis identification of the purified monoclonal antibody E1Y003 are presented.

[0053] Figure 5 The identification results of the E1Y003 monoclonal antibody subtype are presented.

[0054] Figure 6The results demonstrate the detection of E1Y003 specifically binding to CD2v SD1 using flow cytometry and Western blot. In this paper, A represents the flow cytometry results and B represents the Western blot results.

[0055] Figure 7 The binding of E1Y003 and ASFV virus to infected cells is shown. In this paper, A represents the results of immunohistochemical staining, and B represents the results of Western blot analysis.

[0056] Figure 8 The results demonstrate the specificity and potency of the genetically engineered antibody binding assay. In this assay, A represents the specificity assay result, and B represents the potency assay result. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0058] Abbreviations IMGT: The numbering system based on the international ImMunoGeneTics information system® (IMGT) initiated by Lefranc et al. See Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0059] Kabat: An immunoglobulin matching and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0060] Chothia: An immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0061] AbM: The CDR definition method is derived from Martin's related research (Martin AC, Cheetham JC, ReesAR (1989). Modeling antibody hypervariable loops: A combined algorithm. ProcNatl Acad Sci USA 86:9268–9272).

[0062] Contact: The CDR definition method is derived from Martin's related research (Martin, ACR (2001). Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Lab Manuals. Springer, Berlin, Heidelberg.).

[0063] ELISA: Enzyme-linked immunosorbent assay.

[0064] PCR: Polymerase chain reaction.

[0065] HRP: Horseradish peroxidase.

[0066] CDR-H1: Complementarity-determining region 1 in the variable region of the immunoglobulin heavy chain.

[0067] CDR-H2: Complementarity-determining region 2 in the variable region of immunoglobulin heavy chain.

[0068] CDR-H3: Complementarity-determining region 3 in the variable region of the immunoglobulin heavy chain.

[0069] CDR-L1: Complementarity-determining region 1 in the variable region of the immunoglobulin light chain.

[0070] CDR-L2: Complementarity-determining region 2 in the variable region of the immunoglobulin light chain.

[0071] CDR-L3: Complementarity-determining region 3 in the variable region of the immunoglobulin light chain.

[0072] Definitions Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0073] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0074] As used in this article, "antibody" refers to a globulin produced by plasma cells, which are formed from the proliferation and differentiation of B lymphocytes in response to antigen stimulation. Antibodies specifically bind to the corresponding antigens and mediate immune effects. They are mainly found in serum and body fluids and are important immune molecules mediating humoral immunity. Antibodies can encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies), single-chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulin (Ig).

[0075] As used herein, the term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that, apart from possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies within the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0076] As used herein, the term "multispecific antibody" is used in its broadest sense to encompass antibodies exhibiting multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and trispecific antibodies, antibody fragments covalently or non-covalently linked, etc.

[0077] The terms “full-length antibody” and “intact antibody” as used herein are used interchangeably to refer to antibodies that are structurally similar to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions, CH). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant region). The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: the κ (kappa) light chain and the λ (lambda) light chain.

[0078] Within the light and heavy chains, variable and constant regions are linked by a "J" region containing approximately 12 or more amino acid residues, and the heavy chain also contains a "D" region containing approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0079] The term "Fd fragment" as used herein refers to an antibody fragment consisting of VH and CH1 domains. The term "dAb fragment" as used herein refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)). The term "Fab fragment" as used herein refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains. The term "F(ab')2 fragment" as used herein refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region. The term "Fab' fragment" as used herein refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of VH and CH1 domains). The term "Fab'-SH" as used herein refers to a Fab fragment containing free thiol groups.

[0080] As used in this article, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0081] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0082] As used herein, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable regions (VH and VL) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR1-4) and three hypervariable regions (HVR1-3), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain is sufficient to confer antigen-binding specificity. The three HVRs within the VH and VL together constitute the antigen-binding site of Ig, which can bind complementary to the corresponding antigenic epitope; therefore, the HVRs are also called complementarity-determining regions (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The VH or VL chain of the antibody may further contain all or part of the constant regions of the heavy or light chain.

[0083] The CDR of the antibody or antigen-binding fragment thereof disclosed herein can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof disclosed herein is preferably determined by the IMGT, Kabat, Contact, Chothia, or AbM numbering system.

[0084] As used in this article, the term "variable" refers to the fact that certain segments of the variable region are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable region, but is concentrated in three segments called hypervariable regions (HVRs) within the variable regions of the light and heavy chains. The relatively highly conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are tightly held together by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site. Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0085] Antibody "classes" refer to the types of constant structural domains or constant regions possessed by the antibody's heavy chain. Based on differences in heavy chain structure and antigenicity, they can be classified into five classes: μ chain, γ chain, α chain, δ chain, and ε chain. Immunoglobulins composed of different heavy and light chains are respectively called IgA, IgD, IgE, IgG, and IgM. Even within the same class of Ig, the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain differ, thus further subdividing the same class of Ig into different subclasses. For example, human IgG can be divided into IgG1–IgG4; IgA can be divided into IgA1 and IgA2. Based on differences in light chain structure and antigenicity, immunoglobulin (Ig) light chains are divided into κ (kappa) chains and λ (lambda) chains, thus classifying Ig into two types: κ type and λ type.

[0086] "Porcine-derived antibodies" comprise amino acid residues from non-porcine HVRs and amino acid residues from porcine FRs. In some embodiments, porcine-derived antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-porcine antibody, and all or substantially all FRs correspond to the FRs of the porcine antibody. Porcine-derived antibodies may optionally comprise at least a portion of the antibody constant region derived from a porcine antibody. Antibodies in a "porcine-derived form," such as non-porcine antibodies, refer to antibodies that have undergone porcine-derived processing.

[0087] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0088] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody that retains the antibody's antigen-binding activity. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0089] As used herein, the terms "antigen-binding domain" or "antigen-binding site" refer to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0090] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformation composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used as antigens in this invention can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Antigens can also be human proteins, or antigens can be "full-length," unprocessed proteins, and any form of protein produced by intracellular processing, or naturally occurring protein variants, such as splice variants or allelic variants.

[0091] The specific “binding strength” or “affinity” of an antibody or its antigen-binding fragment to an antigen refers to the strength of the non-covalent interaction between a single binding site and its binding ligand (e.g., antigen), and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. Binding affinity is typically expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (Kd and Ka, respectively). In some embodiments, the dissociation constant (Kd) of the molecule binding to the antigen is ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10). -7 M or lower, such as 10 -7 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0092] As used herein, the term "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its natural environment. In this invention, the recombinant polynucleotide encoding an antibody or its antigen-binding fragment contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids of this disclosure further include synthetically generated molecules of this type. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators.

[0093] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably to refer to a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can be transcribed into a large amount of stable mRNA. Once the expression vector is in the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette containing a polynucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof. The term "expression cassette" of the present invention refers to a recombinant or synthetically generated polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof.

[0094] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the bispecific antigen-binding molecule of this invention. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues. Host cells do not refer to propagation material in this document.

[0095] As used herein, the term "pharmaceutical composition" refers to a composition containing one or more antibodies of the present invention or antigen-binding fragments thereof, or a mixture of the present invention with other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0096] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0097] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, such as one comprising any of the antibodies or antigen-binding fragments thereof or the fusion proteins or pharmaceutical compositions disclosed herein, to a subject who has one or more infectious swine diseases or symptoms caused by African swine fever virus (ASFV), and the therapeutic agent has a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in the treated individual or population in an amount that effectively alleviates one or more diseases or symptoms, to induce the regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0098] As used in this article, “prevention” refers to delaying, suppressing, or preventing the onset of infectious diseases in pigs caused by African swine fever virus (ASFV) in subjects.

[0099] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0100] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0101] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0102] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0103] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0104] Sequence information The sequence information involved in the embodiments of the present invention is shown in Table 1: Table 1: Sequence Information

[0105] In the nucleotide sequences of the primers in the table, R represents A or G, Y represents C or T, K represents G or T, M represents A or C, S represents G or C, and W represents A or T.

[0106] Example 1: Expression of CD2v protein 1. Construction of recombinant plasmids expressing CD2v Based on the CD2v gene sequence of the Chinese prevalent ASFV strain Pig / HLJ / 2018 (GenBank: MK333180.1) in GenBank, after sequence analysis, the extracellular segment of CD2v was selected as a template for gene synthesis after codon optimization. Primers were designed to amplify the full-length extracellular segment of CD2v (FL: 18-204AA) and the two IG domains (first domain SD1: 18-103AA; second domain SD2: 117-204AA) via PCR. A signal peptide (SP) and a his tag sequence were added to the N-terminus of each domain, and the amplified sequences were then cloned into the eukaryotic expression vector pCMV. The nucleotide sequence of SP-his-CD2v FL is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2. The nucleotide sequence of SP-his-CD2v SD1 is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4. The nucleotide sequence of SP-his-CD2v SD2 is shown in SEQ ID NO:4. As shown in NO:5, the amino acid sequence is as shown in SEQ ID NO:6. An expression vector in the form of "SP-his-CD2v FL\SD1\SD2" is constructed. After transfection into cells, the expressed protein will cleave the signal peptide to form a mature recombinant protein that is secreted into the cell culture medium.

[0107] 2. Expression of CD2v recombinant protein 1) Preparation of transfection complex Preparation method of PEI transfection reagent solution: Add 100 mg of linear PEI transfection reagent (Mw 40000; LABLEAD, catalog number P4000) to 90 mL of Milli-Q ultrapure water, stir until completely dissolved, adjust the pH to 6.9-7.1, and make up to 100 mL with Milli-Q ultrapure water. Then filter through a 0.22 μm filter membrane and collect the filtrate.

[0108] Preparation of transfection complex (per 1L of cell suspension): Add 1mg of recombinant plasmid (pCMV-SP-his-CD2v FL\SD1\SD2) to 5mL of Hi-exp medium (Opmai, catalog number AC601501), mix well by pipetting, and this is liquid phase A; add 3mL of PEI transfection reagent solution to 5mL of Hi-exp medium, mix well by pipetting, and this is liquid phase B; add liquid phase B to liquid phase A, mix well by pipetting, and then incubate at room temperature for 5min.

[0109] 2) Preparation of cell suspension Collect 293F cells from the culture, count the cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend in Hi-exp medium to achieve a cell concentration of 1×10⁻⁶ cells / mL. 6 Cells / mL.

[0110] 3) Add the transfection complex dropwise to 1L of cell suspension and incubate with shaking at 130rpm for 4-5 days (environmental conditions: 37℃, 8% CO2). Then centrifuge at 4000rpm for 20min, collect the supernatant, and filter it through a 0.45μm filter membrane and collect the filtrate.

[0111] 3. Purification of recombinant proteins Column chromatography was used (column volume 10 mL; packing material: Ni Sepharose 6FF, Cytiva (GE Life), catalog number 17531801). First, the column was equilibrated with equilibration buffer (1×PBS buffer; 300 mM NaCl; pH 7.4). Then, the supernatant obtained from expression in step 2 was loaded onto the column (loading volume 1 L). 5-10 column volumes were loaded with washing buffer (1×PBS buffer; 300 mM NaCl; 50 mM imidazole; pH 7.4). Elution with elution buffer (1×PBS buffer; 300 mM NaCl; 500 mM imidazole; pH 7.4) yielded CD2v FL, SD1, and SD2 proteins.

[0112] 4. Identification of recombinant proteins The concentration of recombinant protein was determined using a nanodrop, and the recombinant protein was identified by SDS-PAGE. Under denaturing conditions, 1-2 μg of recombinant protein sample was loaded onto a 12% SDS-PAGE gel. After electrophoresis, Coomassie Brilliant Blue staining was performed, followed by destaining until clear protein bands were visible. The results are shown below. Figure 1 As shown, since CD2v FL, SD1 and SD2 recombinant proteins are all glycosylated, they exhibit broad bands, and the purified proteins have high purity.

[0113] 5. Binding of recombinant protein to African swine fever-positive serum (ELISA method) Take CD2v FL, SD1, and SD2 recombinant proteins respectively, and dilute them with PBS buffer to a protein concentration of 1 μg / mL to obtain the coating solution. PBST solution: PBS buffer containing 0.05% (v / v) Tween-20. Blocking solution: PBST solution containing 0.2 g / 100 mL BSA. Antibody dilution preparation method: African swine fever positive serum (China Institute of Veterinary Drug Control) was used as positive serum (PC), and serum from unimmunized normal pigs was used as negative control (NC). The serum was diluted 1000-fold with blocking solution.

[0114] Follow these steps to perform the test: 1) Take a 96-well microplate, add coating buffer (100 μL / well), incubate at 4°C for 16 hours (overnight), discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0115] 2) Take the 96-well plate after completing step 1), add blocking solution (200 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0116] 3) Take the 96-well plate from step 2), add antibody dilution buffer (100 μL / well), incubate at room temperature for 1 hour, discard the supernatant, wash 3 times with PBST solution, and pat dry.

[0117] 4) Take the 96-well plate from step 3), add HRP-labeled goat anti-pig IgG antibody (Sigma, AP166P) (100 μL / well, diluted 10,000 times), incubate at room temperature for 1 hour, discard the supernatant, wash 5 times with PBST solution, and pat dry.

[0118] 5) Take the 96-well plate after completing step 4), add TMB colorimetric solution (100 μL / well), and react in the dark for 5-10 minutes.

[0119] 6) Take the 96-well plate from step 5), add 2M sulfuric acid solution (50 μL / well), and then measure the absorbance (OD) at 450 nm. 450 ).

[0120] CD2v FL, SD1, and SD2 recombinant proteins can all bind to African swine fever-positive serum, as shown in the following results. Figure 2 It can be inferred that the recombinant protein and the native conformation of CD2v in ASFV are similar.

[0121] Example 2: Hybridoma Monoclonal Antibody Screening 1. Immunizing mice SPF-grade 6-8 week old Balb / c mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were immunized with CD2vSD1 recombinant protein as the immunogen, following the procedure below: Day 1: First immunization, multiple subcutaneous injections of immunizing agent (composed of 10 μg of immunogen and Freund's complete adjuvant (Sigma, F5881)); Day 29: Second immunization, multiple subcutaneous injections of immunizing agent (composed of 10 μg immunogen and Freund's incomplete adjuvant (Sigma, F5506)); Day 57: Third immunization, multiple subcutaneous injections of immunizing agent (composed of 10 μg of immunogen and Freund's incomplete adjuvant); Day 64: Orbital venous blood was collected, serum was separated and used as test antibodies. The titer was detected by ELISA. Mice with high titers were selected for the fourth immunization, and 10 μg of immunogen was injected intraperitoneally.

[0122] 2. Hybridoma cell fusion and screening 1) Preparation of feeder cells The day before fusion, well-developed Balb / c mice were euthanized by cervical dislocation and sterilized in 75% ethanol aqueous solution. Using a pre-chilled syringe, 8-10 mL of pre-chilled 0.34M sucrose aqueous solution was injected through the lower right peritoneum of the mouse, ensuring the needle did not exit the peritoneum. The peritoneal cavity was massaged with a finger for approximately 1 minute. The fluid from the peritoneal cavity was then aspirated and added to a pre-chilled 50 mL centrifuge tube. Pre-chilled 1640 complete culture medium was added, and the tube was centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 1640 complete culture medium containing HAT (Sigma, HO262) (HAT medium) to a cell concentration of 1×10⁻⁶. 5 Cells / mL were collected, and then the cell suspension was added to a 96-well cell culture plate (100 μL / well) and cultured.

[0123] 2) Preparation of spleen cells Four days after the fourth immunization in step 1, mice were euthanized by enucleation, and blood and spleen were collected separately. Serum was isolated from the blood as an immunopositive control. A 10 cm diameter culture dish was prepared, and 10 mL of 1640 complete medium and DNase (final concentration 5 μg / mL) were added. Mouse spleen was then added, and the mixture was ground and pipetted to form a single-cell suspension. The suspension was filtered through a 70 μm filter and collected into a 50 mL centrifuge tube. The cells were centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cell pellet was resuspended in ACK erythrocyte lysis buffer and incubated at room temperature for 2 min. Cells were washed with 1640 complete medium, centrifuged, and the supernatant was discarded. The cells were then resuspended in 20 mL of 1640 complete medium, mixed thoroughly, and counted.

[0124] 3) Cell fusion Collect healthy SP2 / 0 mouse myeloma cells in the logarithmic growth phase and wash with 1640 complete medium. Mix SP2 / 0 mouse myeloma cells and spleen cells at a ratio of 1:1 to 1:3, centrifuge at 1500 rpm for 10 min, and discard the supernatant. Pat the cells to form a paste, then place in a 37°C water bath. Add 1 mL of preheated 50% PEG solution dropwise, followed by 40 mL of preheated 1640 complete medium. Centrifuge at 1500 rpm for 10 min, and discard the supernatant. Break up the cell pellet, add 10 mL of HAT medium and agitate several times, then add more HAT medium to approximately 90 mL. Mix well and drop the mixture into feeder cell culture plates, 2 drops per well, and incubate at 37°C.

[0125] 4) Screening of hybridoma cell positive wells Four days after cell fusion, the medium was replaced with half of the HAT medium. After approximately 7-10 days, the hybridoma cell clusters reached a certain size. One day before testing, about 200 μL of medium was aspirated and replaced with 200 μL of fresh 1640 complete medium containing HT (Sigma, H0137) (HT medium). On the day of testing, the culture medium from the hybridoma cell clusters was used as the test antibody. Immunopositive serum was used as a positive control, and non-immunized mouse serum was used as a negative control. Binding to CD2v SD1 protein was detected using ELISA to screen for positive clones.

[0126] 5) Subcloning of positive hybridoma cells The first subcloning was performed using HT medium. Positive cloning wells were selected for the first subcloning, and cell state and cell cluster size were observed under a microscope. Under aseptic conditions, the cells to be subcloned were gently pipetted to mix, avoiding air bubbles. 10 μL of the cell count was aspirated, and 10 μL of 0.04% trypan blue solution was added. After mixing, the cells were counted in 8 large squares, and the cell concentration was calculated. Based on the cell count results, 100-150 cells were added to 9.5 mL of medium using the limiting dilution method. After mixing, the mixture was added dropwise to feeder cell culture plates cultured for 1 day.

[0127] After about 5 days of subcloning, the hybridoma clones in each well are counted under an inverted microscope. Once the hybridoma cells have grown to a suitable size, they are tested again and positive single clones are screened for a second subcloning. The culture medium is then replaced with 1640 complete medium. Subcloning is repeated 2-3 times until the obtained single-clone hybridoma cell line can stably secrete the required antibody.

[0128] 6) Expansion culture and cryopreservation of hybridoma cells ① The positive hybridoma cells obtained after identification were expanded and transferred to 24-well cell culture plates and cultured until the confluence reached about 80%.

[0129] ② After completing step ①, transfer the cells to a T25 cell culture flask and culture until the cell confluence reaches approximately 80%.

[0130] ③ After completing step ②, repeatedly blow the culture flask with culture medium, then transfer it to a sterile centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells with 3 mL of cell cryopreservation solution, mix thoroughly, transfer the cell suspension to a cryopreservation tube, transfer it to a programmed cooling box, incubate at -80℃ for 24 h, and then transfer it to liquid nitrogen for long-term storage.

[0131] Based on the above steps, hybridoma cells secreting target monoclonal antibodies (i.e., monoclonal antibodies that specifically bind to CD2v SD1 protein) were obtained, including the monoclonal E1Y003 (derived from the 7B11 clone) contained in this invention. Figure 3 ).

[0132] Example 3: Preparation and Identification of Monoclonal Antibodies 1. Preparation of ascites 1) On day 0, NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were intraperitoneally injected with 500 μL of liquid paraffin (preferably multiparous mice or larger individuals). 2) On days 9-11, collect hybridoma cells in the logarithmic growth phase into centrifuge tubes, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, gently tap the cells, add 40 mL of PBS to wash the cells, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, repeat the washing once, count the cells, and add PBS to prepare 1×10⁻⁶ cells / tubes. 6 -2×10 6 A cell suspension of 1 cell / mL was administered intraperitoneally to each mouse, with a dose of 500 μL.

[0133] 3) Around day 19-23, depending on the condition of the mice, remove the ascites fluid, centrifuge at 12000 rpm for 10 minutes, aspirate the pale yellow ascites fluid, and store at -80℃ for later use.

[0134] 2. Antibody purification Antibodies were purified from ascites fluid using an affinity chromatography column (packing material: protein At Beads LX, Changzhou Tiandi Renhe Biotechnology Co., Ltd., product catalog number: SA08501L).

[0135] 1) First, equilibrate the chromatography column with PBS buffer.

[0136] 2) Then take out the ascites fluid, centrifuge at 12000 rpm for 10 minutes, take the supernatant, dilute it 5 times with PBS buffer, filter it at 0.45 μm, and load the filtered ascites fluid dilution onto the chromatography column.

[0137] 3) After all the ascites fluid has been filtered out, wash with PBS buffer for 10-15 column volumes.

[0138] 4) Elute with glycine buffer (pH 2.5-3.0, 0.1M) and collect the post-column solution. Mix 10 volumes of the post-column solution with 1 volume of neutralization buffer (i.e., pH 9.0, 1M Tris-HCl buffer) to obtain a mixture.

[0139] 5) Subsequently, a 30K ultrafiltration tube (Millipore, UFC903096) was used for concentration and buffer replacement, replacing the buffer system with PBS buffer to obtain the purified monoclonal antibody. The SDS-PAGE electrophoresis image is shown below. Figure 4 .

[0140] 3. Identification of monoclonal antibody subtypes Dilute E1Y003 monoclonal antibody to a working solution of 1 μg / mL. Identify the antibody subtype using a monoclonal antibody subtype identification kit (Beijing Sinocare Technology Co., Ltd., product catalog number SEK003) and follow the instructions.

[0141] See results Figure 5 E1Y003 is the IgG2a subtype.

[0142] Example 4: Specific binding of monoclonal antibody to CD2v protein 1. To enable flow cytometry detection of monoclonal antibody-protein binding, a transmembrane region (TM) and an EGFP sequence were added to the C-terminus of CD2v. The nucleotide sequence of the transmembrane region is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8. The nucleotide sequence of the GFP is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10. Recombinant plasmids for cell membrane expression of FL, SD1, and SD2 were constructed, with the form "SP-his-CD2v FL\SD1\SD2-TM-GFP". After transfection into cells, the recombinant proteins expressed will have their signal peptide cleaved and will be expressed on the cell membrane. The extracellular portion of the protein will be the corresponding extracellular segment of CD2v, and the intracellular portion will be GFP.

[0143] 2. The membrane expression vectors constructed above were transfected into 293T cells (6 cm dish) using PEI transfection reagent. 24 h post-transfection, cells were digested with 2 mM EDTA to form a single-cell suspension. 2 mL of FACS buffer was added, and the cells were washed by centrifugation at 1500 rpm for 5 min, discarding the supernatant. The cells were divided into two aliquots: one aliquot was added to FACS buffer (1×PBS + 0.2% BSA + 2 mM EDTA), and the other aliquot was lysed with RIPA and then boiled in 5×SDS loading buffer (containing 5% β-mercaptoethanol) at 100°C for 10 minutes.

[0144] 3. Add FACS buffer to the cells, centrifuge at 1500 rpm for 5 min to wash the cells, and discard the supernatant. Add 100 μL of E1Y003 diluted to 1 μg / mL with FACS buffer and incubate on ice for 30 min. Wash the cells once with FACS buffer, centrifuge and discard the supernatant. Add 100 μL of 700-fold diluted PE-goat anti-mouse IgG (BioLegend, 405307), and stain on ice in the dark for 30 min. Wash the cells once with FACS buffer, centrifuge and discard the supernatant. Add 400 μL of 2% PFA to each sample to resuspend the cells, and analyze the samples using flow cytometry. The results are analyzed using FlowJo software.

[0145] The results are as follows Figure 6 As shown in Figure A, his-CD2v FL-GFP, his-SD1-GFP, and his-SD2-GFP proteins were expressed on the cell membrane of transfected cells, respectively. The E1Y003 antibody specifically bound CD2v FL and SD1, but could not bind to SD2.

[0146] 4. Run SDS-PAGE electrophoresis on the high-temperature denatured sample prepared in step 2. After transferring the protein to a PVDF membrane, block it at room temperature for 1 hour with blocking buffer (5g skim milk powder dissolved in 100mL TBST buffer, TBST buffer: 1× TBS + 0.1% Tween 20). Add E1Y003 antibody solution diluted to 1μg / mL with blocking buffer and incubate on a shaker at room temperature for 1-2 hours or overnight at 4°C. Wash thoroughly three times with TBST on a shaker at room temperature for 10 minutes each time. Dilute HRP-labeled goat anti-mouse IgG (Zhongshan Jinqiao ZB-2305) with blocking buffer at a ratio of 1:10000 and incubate on a shaker at room temperature for 1 hour. Wash thoroughly three times with TBST buffer on a shaker at room temperature for 10 minutes each time. Develop the PVDF membrane.

[0147] The results are as follows Figure 6As shown in Figure B, E1Y003 can also be used for Western blotting detection. It also specifically binds to CD2v FL and SD1, but not to SD2.

[0148] Example 5: Binding of E1Y003 monoclonal antibody to ASFV virus-infected cells 1. Porcine alveolar macrophages (PAM cells, prepared and provided by Jinyu Baoling Biopharmaceutical Co., Ltd.) infected with ASFV (HLJ / 18) for 72 h were collected in 96-well plates. Uninfected PAM cells were used as a negative control. Immunohistochemical staining was performed. The specific staining steps are as follows: 1) Discard the liquid in the 96-well plate, add 200 μL / well of 10% formalin neutral fixative, and fix at room temperature for 30 min; discard the liquid in the 96-well plate, add 200 μL / well of permeation buffer (500 mL PBS + 5 mL Triton X-100), wash twice, and soak for 5 min each time.

[0149] 2) Discard the liquid in the 96-well plate, fill the cell wells with antigen retrieval solution (10.5g citric acid + 14.19g disodium hydrogen phosphate diluted to 500mL with purified water), place the plate in a water bath at 60℃, and incubate for 2 minutes after the water boils. Remove the plate and allow it to cool naturally for 10-20 minutes. Discard the liquid in the 96-well plate, add 200μL permeation buffer per well, wash twice, and soak for 5 minutes each time.

[0150] 3) Discard the liquid in the 96-well plate, add 100 μL of peroxidase inhibitor per well, incubate at room temperature for 10 min, discard the liquid in the 96-well plate, add 200 μL of permeation buffer per well, wash twice, soaking for 5 min each time.

[0151] 4) Discard the liquid in the 96-well plate, dilute the E1Y003 monoclonal antibody to 40 μg / mL with PBS, add 100 μL / well, add to the 96-well plate, incubate at 37℃ for 30 min, discard the liquid in the 96-well plate, add 200 μL / well permeation buffer, wash twice, soaking for 5 min each time.

[0152] 5) Discard the liquid in the 96-well plate, add 100 μL of HRP-labeled goat anti-mouse IgG (Zhongshan Jinqiao ZB-2305) to each well, incubate at 37°C for 30 min, discard the liquid in the 96-well plate, add 200 μL of permeation buffer per well, wash twice, soaking for 5 min each time.

[0153] 6) Discard the liquid in the 96-well plate, add 100 μL of working concentration DAB chromogenic solution (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) per well; develop color at room temperature for 10 min, discard the chromogenic solution, wash twice with 200 μL of PBS per well, do not discard the solution on the last wash; 7) Observe and record, see Figure 7In Figure A, the left image shows uninoculated blank cells with no specific staining; the right image shows cells inoculated with ASFV virus, which exhibit specific staining.

[0154] 2. After lysing the ASFV-infected cells and negative control PAM cells with RIPA, they were subjected to high-temperature denaturation at 100°C for 10 minutes with 5×SDS loading buffer. Western blot verification confirmed that E1Y003 could indeed specifically bind to the corresponding band in ASFV-infected cells, such as... Figure 7 The red arrow in section B shows the CD2v protein and its short fragments after cleavage or degradation.

[0155] Example 6: Obtaining the variable region sequence of the E1Y003 monoclonal antibody Take 1×10 6 -1×10 7 Hybridoma cells of type E1Y003 were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed with PBS and centrifuged again to remove the supernatant. 1 mL of pre-chilled Trizol (Invitrogen, 15596018CN) was added, and RNA was extracted according to the manufacturer's instructions. cDNA was reverse transcribed using oligo dT primers and M-MLV reverse transcriptase (Progema, M1705).

[0156] Using cDNA as a template, primers targeting the antibody variable region were used. The upstream primers for the mouse VH sequence (SEQ ID NO: 11-22 in Table 1) were paired with the downstream primers for the corresponding heavy chain in Table 1 (SEQ ID NO: 23 in Table 1), and the upstream primers for the mouse Vκ sequence (SEQ ID NO: 24-34 in Table 1) were paired with the downstream primers for the light chain in Table 1 (SEQ ID NO: 35 in Table 1). PCR amplification was performed using PrimeSTAR Max DNA polymerase (TaKaRa, R045). The amplification system and PCR reaction program are shown in Tables 2 and 3 below.

[0157] Table 2: PCR amplification system

[0158] Table 3: PCR reaction procedure

[0159] PCR amplification products were analyzed by 1% agarose gel electrophoresis. The specific band of the correct size in the amplification product was excised, the gel product was recovered and sent to a sequencing company for sequencing to obtain the gene sequence of the antibody heavy chain variable region and the gene sequence of the antibody light chain variable region. The amino acid sequence of the antibody heavy chain variable region and the amino acid sequence of the antibody light chain variable region were further obtained.

[0160] The amino acid sequence of the variable region of the heavy chain of the E1Y003 antibody is shown in SEQ ID NO:36, the nucleotide sequence of the variable region of the heavy chain is shown in SEQ ID NO:37, the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:38, and the nucleotide sequence of the variable region of the light chain is shown in SEQ ID NO:39. The CDR amino acid sequence of the E1Y003 antibody is shown in Tables 4 and 5.

[0161] Table 4: CDR-H1, CDR-H2, and CDR-H3 amino acid sequences of E1Y003 antibody

[0162] Table 5: CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of E1Y003 antibody

[0163] Example 7: Preparation of Genetically Engineered Antibodies The signal peptide sequence (amino acid sequence as shown in SEQ ID NO:65, nucleotide sequence as shown in SEQ ID NO:66), the E1Y003 heavy chain variable region sequence, and the mouse heavy chain constant region sequence (mouse heavy chain (IgG2a subtype) constant region amino acid sequence as shown in SEQ ID NO:61, nucleotide sequence as shown in SEQ ID NO:62) were cloned and recombined into the PTT3 vector to obtain the E1Y003 heavy chain recombinant expression plasmid. The signal peptide sequence (amino acid sequence as shown in SEQ ID NO:65, nucleotide sequence as shown in SEQ ID NO:66), the E1Y003 light chain variable region sequence, and the mouse light chain constant region sequence (mouse light chain (Igκ subtype) constant region amino acid sequence as shown in SEQ ID NO:63, nucleotide sequence as shown in SEQ ID NO:64) were cloned and recombined into the PTT3 vector to obtain the E1Y003 antibody light chain recombinant expression plasmid. Following the method described in Example 1, 293F cells were co-transfected with recombinant heavy and light chains at a 1:1 ratio using PEI transfection reagent. After 6 days of culture, antibodies secreted in the culture medium were obtained. The antibody in the culture medium was purified according to the method described in Example 3 to obtain the highly pure genetically engineered antibody enE1Y003. The specificity and antibody titer of enE1Y003 were determined using the ELISA method described above, and the results are shown in [Figure 1]. Figure 8 Figure A shows that enE1Y003 specifically binds to CD2v FL and SD1 proteins, but cannot bind to CD2v SD2. Figure B shows that E1Y003 and enE1Y003, which were serially diluted, began to bind to CD2v SD1 protein at concentrations below 10 ng / mL, and there was no significant difference in titer between the two.

[0164] The results above show that the variable region sequence obtained by E1Y003 can be used to prepare genetically engineered antibodies that bind to the first domain of CD2v.

[0165] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus CD2v protein, wherein, The antibody or its antigen-binding fragment contains the following complementarity-determining region: (a1) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region shown in SEQ ID NO:36; and / or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region shown in SEQ ID NO:38; (a2) The heavy chain variable regions contain CDR-H1, CDR-H2 and CDR-H3, and / or the light chain variable regions contain CDR-L1, CDR-L2 and CDR-L3, wherein, compared with the heavy chain variable regions and / or light chain variable regions described in (a1), at least one CDR contains a mutation, said mutation being a substitution, deletion or addition of one or more amino acids; preferably, said substitution is a conservative substitution.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The complementary decision region is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system; Preferably, the antibody or its antigen-binding fragment includes the following heavy chain variable region and / or light chain variable region, wherein the complementarity-determining region is defined according to the IMGT numbering system: The heavy chain variable region contains the following three CDRs: CDR-H1 with sequence SEQ ID NO:40 or a variant thereof; CDR-H2 with sequence SEQ ID NO:41 or a variant thereof; CDR-H3 with sequence SEQ ID NO:42 or a variant thereof; and / or, the light chain variable region contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:52 or a variant thereof; CDR-L2 with sequence SEQ ID NO:53 or a variant thereof; CDR-L3 with sequence SEQ ID NO:54 or a variant thereof; Preferably, the antibody or its antigen-binding fragment includes the following heavy chain variable region and / or light chain variable region, wherein the complementarity-determining region is defined according to the Kabat numbering system: The heavy chain variable region contains the following three CDRs: CDR-H1 with sequence SEQ ID NO:43 or a variant thereof; CDR-H2 with sequence SEQ ID NO:44 or a variant thereof; CDR-H3 with sequence SEQ ID NO:45 or a variant thereof; and / or, the light chain variable region contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:55 or a variant thereof; CDR-L2 with sequence SEQ ID NO:56 or a variant thereof; CDR-L3 with sequence SEQ ID NO:57 or a variant thereof; Preferably, the antibody or its antigen-binding fragment includes the following heavy chain variable region and / or light chain variable region, wherein the complementarity-determining region is defined according to the Chothia numbering system: The heavy chain variable region contains the following three CDRs: CDR-H1 with sequence SEQ ID NO:46 or a variant thereof; CDR-H2 with sequence SEQ ID NO:47 or a variant thereof; CDR-H3 with sequence SEQ ID NO:48 or a variant thereof; and / or, the light chain variable region contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:55 or a variant thereof; CDR-L2 with sequence SEQ ID NO:56 or a variant thereof; CDR-L3 with sequence SEQ ID NO:57 or a variant thereof; Preferably, the antibody or its antigen-binding fragment includes the following heavy chain variable region and / or light chain variable region, wherein the complementarity-determining region is defined according to the Contact numbering system: The heavy chain variable region contains the following three CDRs: CDR-H1 with sequence SEQ ID NO:49 or a variant thereof; CDR-H2 with sequence SEQ ID NO:50 or a variant thereof; CDR-H3 with sequence SEQ ID NO:51 or a variant thereof; and / or, the light chain variable region contains the following three CDRs: CDR-L1 with sequence SEQ ID NO:58 or a variant thereof; CDR-L2 with sequence SEQ ID NO:59 or a variant thereof; CDR-L3 with sequence SEQ ID NO:60 or a variant thereof; The variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment comprises: The heavy chain variable region contains the sequence shown in SEQ ID NO:36 or a variant thereof and / or the light chain variable region contains the sequence shown in SEQ ID NO:38 or a variant thereof; The variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a porcine antibody; Preferably, the antibody is of type IgA, IgD, IgE, IgG or IgM; more preferably, the antibody is of type IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that, The antibody or its antigen-binding fragment further comprises a constant region derived from or originating from mouse immunoglobulins, or a constant region derived from or originating from porcine immunoglobulins. Preferably, the heavy chain of the antibody or its antigen-binding fragment contains a heavy chain constant region derived from or originating from mouse immunoglobulins. Preferably, the antibody or its antigen-binding fragment contains a wild-type Fc region, or contains a mutated or chemically modified Fc region that has altered effector functions compared to the wild-type Fc region; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region derived from or originating from mouse IgG2a; Preferably, the light chain of the antibody or its antigen-binding fragment comprises a light chain constant region derived from or originating from mouse immunoglobulins; Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region derived from or originating from mouse Igκ; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO:61 or a variant thereof, the variant having a conservative substitution of up to 20 amino acids compared to SEQ ID NO:61; Preferably, the antibody or its antigen-binding fragment comprises a light chain constant region as shown in SEQ ID NO:63 or a variant thereof, the variant having a conservative substitution of up to 20 amino acids compared to SEQ ID NO:63; Preferably, the antibody or its antigen-binding fragment comprises a heavy chain constant region as shown in SEQ ID NO:61 and a light chain constant region as shown in SEQ ID NO:

63.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that, The antibody or its antigen-binding fragment comprises: The heavy chain including the heavy chain variable region shown in SEQ ID NO:36 and the heavy chain constant region shown in SEQ ID NO:61, and the light chain including the light chain variable region shown in SEQ ID NO:38 and the light chain constant region shown in SEQ ID NO:

63.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, The antibody or its antigen-binding fragment is ScFv, Fab, Fab', Fab'-SH, F(ab')2, Fv fragment, disulfide-linked Fv, biantibody, bispecific antibody or multispecific antibody; Preferably, the antibody or its antigen-binding fragment is labeled; Preferably, the antibody or its antigen-binding fragment carries a detectable label.

8. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, its heavy chain and / or light chain, and its heavy chain variable region and / or light chain variable region; Preferably, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding the antibody heavy chain variable region and / or a nucleic acid molecule encoding the antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region comprises: (i) the nucleotide sequence shown in SEQ ID NO:37, (ii) a sequence substantially identical to SEQ ID NO:37, or (iii) a degenerate sequence of (i) or (ii) above; and / or, the nucleic acid molecule encoding the antibody light chain variable region comprises: (iv) the nucleotide sequence shown in SEQ ID NO:39, (v) a sequence substantially identical to SEQ ID NO:39, or (vi) a degenerate sequence of (iv) or (v) above.

9. A vector comprising the nucleic acid molecule of claim 8; preferably, the vector is a cloning vector or an expression vector.

10. A host cell comprising the nucleic acid molecule of claim 8 or the vector of claim 9.

11. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising culturing a host cell according to claim 10 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

12. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, or a nucleic acid molecule as described in claim 8, or a carrier as described in claim 9, or a host cell as described in claim 10, and a pharmaceutically acceptable carrier and / or excipient.

13. A detection kit containing an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, or a nucleic acid molecule as described in claim 8, or a vector as described in claim 9, or a host cell as described in claim 10, the kit being used to detect the presence or level of ASFV or a protein containing the first extracellular domain SD1 of the CD2v protein of African swine fever virus in a sample.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the nucleic acid molecule according to claim 8, or the carrier according to claim 9, or the host cell according to claim 10, or the pharmaceutical composition according to claim 12, in the preparation of a reagent, wherein the reagent is used for one or more of the following purposes: 1) Detect the presence or level of African swine fever virus or protein containing the first extracellular domain SD1 of the African swine fever virus CD2v protein in the sample; 2) Prevention and / or treatment of diseases associated with African swine fever virus; Preferably, the disease related to the African swine fever virus is African swine fever.

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