Bispecific antibody targeting African swine fever virus and hog cholera virus and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a bispecific antibody targeting African swine fever virus and hog cholera virus and application of the bispecific antibody, the bispecific antibody shows high binding capacity to p30 protein of the African swine fever virus and E2 protein of the hog cholera virus, and it is indicated that the bispecific antibody can recognize the African swine fever virus and the hog cholera virus at the same time. When the bispecific antibody disclosed by the invention is used for preparing a detection product, double-flux detection of screening African swine fever viruses and swine fever viruses can be realized at the same time through one-time detection, and a new thought is provided for multiple detection of swine disease serology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a bispecific antibody targeting African swine fever virus and classical swine fever virus and application thereof. BACKGROUND

[0002] African swine fever (ASF) is a viral infectious disease of pigs, and its pathogen is African swine fever virus (ASFV). At present, there is no vaccine and drug available in China, and the prevention and control of ASF can only rely on reliable diagnostic methods and strict biosafety measures, so the diagnostic method is crucial for the prevention and control of ASF. ASFV is a linear double-stranded DNA virus with a capsid, has a large genome, and can encode more than 160 proteins. Among them, the CP204L gene encodes the p30 protein, which is located in the inner membrane of ASFV. The protein is expressed in the early stage of viral infection and is one of the most abundant and immunogenic proteins in the early stage of infection, which can induce a large number of specific antibodies in infected pigs. Years of research have shown that p30 is one of the best antigens for African swine fever virus serological detection. Classical swine fever (CSF) is an acute and severe infectious disease caused by classical swine fever virus (CSFV) and is prevalent worldwide. It has a high morbidity and mortality rate in pig populations. It is listed as a class A disease by the World Organization for Animal Health (OIE). The genome of the classical swine fever virus has only one open reading frame, which can encode four structural proteins and eight non-structural proteins. Among them, the structural proteins E0 and E2 have strong antigenicity and can induce protective immunity in natural hosts, producing neutralizing antibodies, and are the preferred proteins for developing new vaccines and detection kits for classical swine fever.

[0003] Monoclonal antibodies are highly specific antibodies that only act on a single epitope of an antigen, and have been widely used for the detection and treatment of many diseases. Since the concept of bispecific antibodies was proposed in 1960, and with the continuous development of antibody technology in recent years, bispecific antibody technology has become more mature, and its application has become more widespread. Using bispecific antibody technology, specific antibodies that can recognize two viruses at the same time can be constructed. Compared with traditional monoclonal antibodies, this type of bispecific antibody can simultaneously target two target pathogens in one detection, which not only helps to save detection costs, but also significantly improves detection efficiency, achieving time-saving and labor-saving diagnosis. In recent years, with the increasing frequency of global trade and the deepening of international exchanges, the risk of disease transmission has also intensified, and the demand for efficient detection methods, especially "one experiment, multiple screening", has become increasingly urgent. SUMMARY

[0004] To solve one of the above technical problems in the prior art, the present application provides a bispecific antibody targeting African swine fever virus and classical swine fever virus and application thereof.

[0005] The first aspect of the present disclosure provides an antibody or antigen-binding fragment thereof specifically binding to the p30 protein of African swine fever virus, comprising: the VH comprising the 3 CDRs of HCDR1 having the amino acid sequence set forth in SEQ ID NO: 19, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 21; and the VL comprising the 3 CDRs of LCDR1 having the amino acid sequence set forth in SEQ ID NO: 22, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 24; wherein the CDRs are defined according to the IMGT numbering system; or The antibody or antigen-binding fragment thereof comprises: the VH comprising the 3 CDRs of HCDR1 having the amino acid sequence set forth in SEQ ID NO: 25, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 26, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 27; and the VL comprising the 3 CDRs of LCDR1 having the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 24; wherein the CDRs are defined according to the Kabat numbering system; or The antibody or antigen-binding fragment thereof comprises: the VH comprising the 3 CDRs of HCDR1 having the amino acid sequence set forth in SEQ ID NO: 30, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 31, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 27; and the VL comprising the 3 CDRs of LCDR1 having the amino acid sequence set forth in SEQ ID NO: 28, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 29, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 24; wherein the CDRs are defined according to the Chothia numbering system; or The antibody or antigen-binding fragment thereof comprises: the VH comprises the HCDR1 having the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 having the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 having the amino acid sequence set forth in SEQ ID NO: 34; and, the VL comprises the LCDR1 having the amino acid sequence set forth in SEQ ID NO: 35, the LCDR2 having the amino acid sequence set forth in SEQ ID NO: 36, and the LCDR3 having the amino acid sequence set forth in SEQ ID NO: 37; wherein the CDRs are defined according to the Contact numbering system.

[0006] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof further comprises a framework region of the heavy chain variable region.

[0007] In some embodiments, the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region of an immunoglobulin derived from a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose, or a mutant thereof.

[0008] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof further comprises a framework region of the light chain variable region.

[0009] In some embodiments, the framework region of the light chain variable region comprises a framework region of a light chain variable region of an immunoglobulin derived from a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervine, mink, chicken, duck, or goose, or a mutant thereof.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence 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%, or at least 99% sequence identity thereto; and, a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence 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%, or at least 99% sequence identity thereto.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region.

[0012] In some embodiments, the heavy chain constant region comprises at least a portion of a heavy chain constant region of an immunoglobulin derived from a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervid, marten, chicken, duck, or goose, or a mutant thereof. In some embodiments, the heavy chain constant region comprises at least a portion of a heavy chain constant region of an immunoglobulin derived from a murine, primate, bovine, equine, porcine, ovine, caprine, canine, feline, leporine, camelid, donkey, cervid, marten, chicken, duck, or goose, or a mutant thereof.

[0013] In some embodiments, the heavy chain constant region comprises a heavy chain constant region derived from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM immunoglobulin.

[0014] In some embodiments, the heavy chain constant region comprises an amino acid sequence set forth in SEQ ID NO: 66, or an amino acid sequence 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%, or at least 99% sequence identity thereto.

[0015] In some embodiments, the heavy chain constant region comprises a heavy chain constant region derived from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM immunoglobulin.

[0016] In some embodiments, the heavy chain constant region comprises an amino acid sequence set forth in SEQ ID NO: 66, or an amino acid sequence 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%, or at least 99% sequence identity thereto.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof is a murine, chimeric, humanized, or fully human antibody.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof comprises a monoclonal antibody, a nanobody, a Fab fragment, a Fab’ fragment, a Fab’-SH fragment, a F(ab’)2 fragment, a Fv fragment, a single-chain Fv (scFv), a dsFv, or a Fd fragment.

[0019] A second aspect of the present disclosure provides a bispecific antibody comprising two protein functional regions targeting antigens, wherein one protein functional region comprises the antibody or antigen-binding fragment thereof specifically binding to the p30 protein of the African swine fever virus according to the first aspect of the present disclosure.

[0020] In some embodiments, the bispecific antibody includes a first protein functional region that specifically binds to the African swine fever virus p30 protein and a second protein functional region that specifically binds to the swine fever virus E2 protein. The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 42, HCDR2 having the amino acid sequence shown in SEQ ID NO: 43, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 44; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 45, LCDR2 having the amino acid sequence shown in SEQ ID NO: 46, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; The CDR is defined according to the IMGT numbering system; or, The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 48, HCDR2 having the amino acid sequence shown in SEQ ID NO: 49, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 50; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 51, LCDR2 having the amino acid sequence shown in SEQ ID NO: 52, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; The CDR is defined according to the Kabat numbering system; or, The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 30, HCDR2 having the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 53, HCDR2 having the amino acid sequence shown in SEQ ID NO: 54, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 50; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 51, LCDR2 having the amino acid sequence shown in SEQ ID NO: 52, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; The CDR is defined according to the Chothia numbering system; or, The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 32, HCDR2 having the amino acid sequence shown in SEQ ID NO: 33, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 34; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 35, LCDR2 having the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 37; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 55, HCDR2 having the amino acid sequence shown in SEQ ID NO: 56, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 57; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 58, LCDR2 having the amino acid sequence shown in SEQ ID NO: 59, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 60; The CDR is defined according to the Contact numbering system.

[0021] In some embodiments, the heavy chain variable region of the first protein functional region further includes the framework region of the heavy chain variable region.

[0022] In some embodiments, the heavy chain variable region of the second protein functional region further includes the framework region of the heavy chain variable region.

[0023] In some embodiments, the light chain variable region of the first protein functional region further includes the framework region of the light chain variable region.

[0024] In some embodiments, the light chain variable region of the second protein functional region further includes the framework region of the light chain variable region.

[0025] In some embodiments, the first protein functional region includes: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence 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%, or at least 99% sequence identity with it; and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 18, or an amino acid sequence 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%, or at least 99% sequence identity with it.

[0026] In some embodiments, the second protein functional region includes: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 40, or an amino acid sequence 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%, or at least 99% sequence identity with it; and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 41, or an amino acid sequence 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%, or at least 99% sequence identity with it.

[0027] In some implementations, the first and second protein functional regions are directly connected or connected through a linker fragment.

[0028] In some implementations, the first protein functional region and the second protein functional region are independently one, two, or more.

[0029] In some embodiments, the first protein functional region and the second protein functional region are each independently an immunoglobulin or an antigen-binding fragment.

[0030] In some embodiments, the antigen-binding fragment includes a Fab fragment, a Fab' fragment, a Fab'-SH fragment, an F(ab')2 fragment, an Fv fragment, or a single-chain Fv (scFv); more specifically, a Fab fragment, an F(ab')2 fragment, or a single-chain Fv (scFv).

[0031] In some embodiments, the first protein functional region is an immunoglobulin and the second protein functional region is an antigen-binding fragment; or, the first protein functional region is an antigen-binding fragment and the second protein functional region is an immunoglobulin.

[0032] In some embodiments, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain Fv.

[0033] In some embodiments, when the first protein functional region is an immunoglobulin, the first protein functional region includes a heavy chain constant region and / or a light chain constant region.

[0034] In some implementations, the single chain Fv includes a heavy chain variable region, a light chain variable region, and a connecting segment for connecting the heavy chain variable region and the light chain variable region.

[0035] In some embodiments, the single-chain Fv has the following structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0036] In some embodiments, the single-chain Fv is connected via a linker fragment to the C-terminus of the heavy chain of the immunoglobulin, the N-terminus of the heavy chain of the immunoglobulin, or the C-terminus of CH1 of the immunoglobulin.

[0037] In some embodiments, the single-chain Fv is attached to the C-terminus of the heavy chain of the immunoglobulin via a linker fragment.

[0038] In some embodiments, the number of immunoglobulins is 1, and the number of single-chain Fvs is 2.

[0039] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain: The first polypeptide chain comprises: the heavy chain of the immunoglobulin, the linker fragment, and the single-chain Fv; the second polypeptide chain comprises: the light chain of the immunoglobulin.

[0040] In some embodiments, the first polypeptide chain comprises amino acids 19-741 of SEQ ID NO: 71; and The second polypeptide chain includes the amino acid sequence from position 19 to 231 of SEQ ID NO: 70.

[0041] A third aspect of this disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as described in the first aspect of this disclosure, or a nucleotide sequence encoding a bispecific antibody as described in the second aspect of this disclosure.

[0042] In some embodiments, the nucleotide sequence of the heavy chain variable region encoding the antibody or its antigen-binding fragment of the first aspect of the invention comprises: the nucleotide sequence shown in SEQ ID NO: 38, or a nucleotide sequence having at least 60%, at least 70%, 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%, or at least 99% sequence identity with it.

[0043] In some embodiments, the nucleotide sequence of the light chain variable region encoding the antibody or its antigen-binding fragment of the first aspect of the invention comprises: the nucleotide sequence shown in SEQ ID NO: 39, or a nucleotide sequence having at least 60%, at least 70%, 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%, or at least 99% sequence identity with it.

[0044] In some embodiments, the nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the first aspect of the present invention further comprises a nucleic acid molecule encoding the heavy chain constant region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention and a nucleic acid molecule encoding the light chain constant region of the antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0045] In some embodiments, the nucleotide sequence encoding the heavy chain constant region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: the nucleotide sequence shown in SEQ ID NO: 65, or a nucleotide sequence having at least 60%, at least 70%, 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%, or at least 99% sequence identity with it.

[0046] In some embodiments, the nucleotide sequence encoding the light chain constant region of the antibody or its antigen-binding fragment of the first aspect of the invention comprises: the nucleotide sequence shown in SEQ ID NO: 67, or a nucleotide sequence having at least 60%, at least 70%, 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%, or at least 99% sequence identity with it.

[0047] This disclosure provides a fourth aspect of a carrier comprising the nucleic acid molecules described in the third aspect of this disclosure.

[0048] In some embodiments, the vector is a cloning vector or an expression vector.

[0049] 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.

[0050] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0051] The fifth aspect of this disclosure provides a host cell comprising the nucleic acid molecule described in the third aspect of this disclosure or the vector described in the fourth aspect of this disclosure.

[0052] In some embodiments, the host cell does not involve reproductive material.

[0053] 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.

[0054] The sixth aspect of this disclosure provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect of this disclosure or the bispecific antibody described in the second aspect of this disclosure, comprising culturing the host cell described in the fifth aspect of this disclosure under conditions that allow expression of the antibody or antigen-binding fragment thereof or bispecific antibody, and recovering the antibody or antigen-binding fragment thereof or bispecific antibody from the cultured host cell culture.

[0055] The seventh aspect of this disclosure provides conjugates comprising the antibody or antigen-binding fragment thereof described in the first aspect of this disclosure, or the bispecific antibody described in the second aspect of this disclosure; and a conjugation portion.

[0056] In some implementations, the coupling portion may include, but is not limited to, detectable markers.

[0057] In some embodiments, the detectable marker 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 markers described above can be linked to the antibodies or antigen-binding fragments of the present invention or multispecific antibodies of different lengths via linkers of varying lengths to reduce potential steric hindrance.

[0058] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0059] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0060] This eighth aspect of the disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the disclosure, or a bispecific antibody as described in the second aspect of the disclosure, or a nucleic acid molecule as described in the third aspect of the disclosure, or a carrier as described in the fourth aspect of the disclosure, or a host cell as described in the fifth aspect of the disclosure, or a conjugate as described in the seventh aspect of the disclosure, and a pharmaceutically acceptable carrier and / or excipient.

[0061] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0062] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0063] The ninth aspect of this disclosure provides a detection product comprising an antibody or antigen-binding fragment thereof as described in the first aspect of this disclosure, or a bispecific antibody as described in the second aspect of this disclosure, or a nucleic acid molecule as described in the third aspect of this disclosure, or a vector as described in the fourth aspect of this disclosure, or a host cell as described in the fifth aspect of this disclosure, or a conjugate as described in the seventh aspect of this disclosure.

[0064] In some embodiments, the detection product includes a detection kit, a detection chip, or a test strip.

[0065] In some embodiments, the test strip is a colloidal gold test strip prepared based on the principle of double-antibody sandwich antigen detection.

[0066] This tenth aspect of the disclosure provides for the use of the antibody or antigen-binding fragment thereof described in the first aspect of the disclosure in any one of c1)-c6): c1) Prepare products for the diagnosis of African swine fever virus infection or the disease it causes; c2) Prepare products for the prevention and / or treatment of African swine fever virus infection or diseases caused by it; c3) Prepare products for detecting the presence or level of African swine fever virus or its p30 protein in samples; c4) Detect the presence or level of African swine fever virus or its p30 protein in the sample; c5) Screening for drugs to prevent and / or treat African swine fever virus infection or the disease it causes; c6) Products for the preparation of drugs for screening the prevention and / or treatment of African swine fever virus infection or diseases caused by it.

[0067] In some implementations, the applications described in c4) and c5) do not involve the diagnosis or treatment of diseases.

[0068] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0069] In some embodiments, the body fluid includes at least one of blood and lymph.

[0070] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0071] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0072] In some implementations, the test subject includes pigs.

[0073] The eleventh aspect of this disclosure provides the use of the bispecific antibody described in the second aspect of this disclosure in any one of c1)-c6): c1) Prepare products for the diagnosis of African swine fever virus and / or classical swine fever virus infection or diseases caused by it; c2) Prepare products for the prevention and / or treatment of African swine fever virus and / or classical swine fever virus infection or diseases caused by it; c3) Prepare products for detecting the presence or level of African swine fever virus, African swine fever virus p30 protein, classical swine fever virus and / or classical swine fever virus E2 protein in samples; c4) Detect the presence or level of African swine fever virus, African swine fever virus p30 protein, classical swine fever virus and / or classical swine fever virus E2 protein in the sample; c5) Screening for drugs to prevent and / or treat African swine fever virus and / or classical swine fever virus infection or the disease it causes; c6) Products for the preparation of drugs for screening the prevention and / or treatment of African swine fever virus and / or classical swine fever virus infection or diseases caused by it.

[0074] In some implementations, the applications described in c4) and c5) do not involve the diagnosis or treatment of diseases.

[0075] In some implementations, the applications described in c4) and c5) do not involve the diagnosis or treatment of diseases.

[0076] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0077] In some embodiments, the body fluid includes at least one of blood and lymph.

[0078] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0079] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0080] In some implementations, the test subject includes pigs.

[0081] The beneficial effects of this invention are: This invention provides an antibody or antigen-binding fragment thereof that specifically binds to the p30 protein of African swine fever virus. The antibody has good binding activity to the p30 protein of African swine fever virus and can be used to prepare products for detecting the presence or level of African swine fever virus or its p30 protein in samples.

[0082] This invention provides a bispecific antibody with specific binding activity against African swine fever virus (ASFV) and classical swine fever virus (CSFV). The bispecific antibody was constructed by fusing single-chain variable fragments of the bispecific antibody using a flexible linker. This bispecific antibody exhibits high binding capacity to both the p30 protein of ASFV and the E2 protein of CSFV, indicating that it can simultaneously recognize both ASFV and CSFV.

[0083] The colloidal gold test strip prepared using the present invention, based on the principle of double antibody sandwich antigen detection, can achieve dual-throughput detection of African swine fever virus and classical swine fever virus in a single experiment, providing a new approach for multiplex detection of swine diseases in serology. Attached Figure Description

[0084] Figure 1 The SDS-PAGE electrophoresis results of p30 protein are shown. A: Elution peak of p30 protein; B: SDS-PAGE detection and identification diagram of p30 protein, 1 is the protein marker; 2 is the p30 protein.

[0085] Figure 2 The results of the reactivity assay for mAbs in ascites fluid are shown. 1 represents the protein marker; 2 represents the p30 recombinant protein; and 3 represents the pET15b empty vector protein.

[0086] Figure 3 This demonstrates that the monoclonal antibody can specifically recognize the p30 protein.

[0087] Figure 4 The diagram shows the construction of the heavy chain recombinant expression plasmid for the monoclonal antibody.

[0088] Figure 5 The diagram shows the construction of the light chain recombinant expression plasmid for the monoclonal antibody.

[0089] Figure 6 The structure of the bispecific antibody is shown.

[0090] Figure 7 The diagram shows the construction pattern of the recombinant plasmid for the bispecific antibody heavy chain.

[0091] Figure 8The results of SDS-PAGE and size exclusion chromatography for the monoclonal antibody are shown. In the figure, A is the SDS-PAGE result of the monoclonal antibody; B is the size exclusion chromatography result of the monoclonal antibody.

[0092] Figure 9 The results of SDS-PAGE and size exclusion chromatography detection of bispecific antibodies are shown: A is the SDS-PAGE detection result of bispecific antibodies; B is the size exclusion chromatography detection result of bispecific antibodies.

[0093] Figure 10 The results of antibody binding activity assays are shown, where A represents the results of monoclonal antibody binding activity assays, and B represents the results of bispecific antibody binding activity assays.

[0094] Figure 11 The results of antibody specificity identification are shown; where A represents the specificity identification result of monoclonal antibodies and B represents the specificity identification result of bispecific antibodies.

[0095] Figure 12 A schematic diagram illustrating the principle of result determination for colloidal gold test strips is shown. Detailed Implementation

[0096] 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.

[0097] 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.

[0098] 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.

[0099] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0100] 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).

[0101] 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.

[0102] 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, trispecific antibodies, antibody fragments covalently or non-covalently linked together, etc.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] The CDR of the antibody or antigen-binding fragment of the present invention 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 of the present invention is preferably determined by the IMGT, Kabat, Contact, Chothia, or AbM numbering system.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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).

[0115] 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.

[0116] 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.

[0117] Sequence information The sequence information involved in the embodiments of the present invention is shown in Table 1.

[0118] Table 1: Sequence Information

[0119] In the primer sequences in the table above, R = A / G, S = C / G, Y = C / T, M = C / A, K = T / G, H = A / T, and D = G / T / A.

[0120] Example 1: Preparation of monoclonal antibodies that specifically bind to the p30 protein of African swine fever virus 1. Obtaining and purifying African swine fever p30 protein 1.1 Primer Design Based on the reference sequence of the p30 gene of HLJ strain 2018 published in GenBank (a total of 585 bp between 124824 and 125408 in GenBank: MK333180.1), amplification primers were designed using Primer 5.0 software, and XhoI and BamHI restriction sites were introduced into the upstream and downstream primers, respectively, and named p30U and p30L.

[0121] The amplified sequence was 585 bp in length. The primers were synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0122] 1.2 Construction of recombinant expression plasmid and expression and purification of p30 protein The target sequence was codon-optimized and cloned into the vector pET15b to obtain the recombinant plasmid pET15b-p30. This plasmid was transformed into E. coli BL21(DE3), and single colonies were picked and cultured in LB medium containing ampicillin (final concentration 50 μg / mL) at 37°C and 225 rpm on a shaker until the logarithmic growth phase (OD200). 600 When the concentration of the compound p30 protein was 0.6–1.0, IPTG was added and the mixture was incubated at 16°C overnight to induce expression of the target protein. The expressed recombinant p30 protein was purified using standard purification methods with Ni-NTA agarose resin. Finally, the target protein was eluted with 250 mmol / L imidazole and purified with 10 kJ of iodine. Da Concentrate the elution buffer using ultrafiltration, replace the protein buffer with PBS, and perform SDS-PAGE analysis on 10 μL of the protein solution. SDS-PAGE electrophoresis results (…) Figure 1 The results showed that the purified target protein was 24 kDa and appeared as a single band, indicating high protein purity. The protein concentration was determined to be 2.5 mg / mL using a BCA protein assay kit. The p30 protein was aliquoted and stored at -80°C for later use.

[0123] The p30 target gene sequence was cloned into the pFastBac1 vector using the same method to construct the pFastBac1-ASFV-p30 recombinant plasmid. The correctly sequenced positive recombinant plasmid pFastBac1-ASFV-p30 was transformed into DH10Bac competent cells, and blue-white screening was performed. After culturing at 37℃ for 48 h, white colonies were selected and identified using the M13 universal primers. The cells were then expanded and cultured. Subsequently, the recombinant plasmid was extracted using a plasmid extraction kit and named Bacmid-ASFV-p30 for use in recombinant baculovirus.

[0124] 2. Preparation and screening of p30 monoclonal antibodies 2.1 Establishment of Indirect ELISA Detection Method The optimal working concentrations of coating antigen and antibody for indirect ELISA were determined using a square matrix titration method. Purified p30 antigen was diluted to 1 mg / mL and then diluted with coating buffer (0.05 mol / L, pH 9.6 carbonate buffer) at dilutions of 1:400, 1:600, 1:800, 1:1000, 1:1200, and 1:1400, respectively. 100 μL of the antigen dilution was then applied to each well of the ELISA plate and incubated overnight at 4°C. BALB / c mouse positive and negative sera were serially diluted at 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200, respectively. SP2 / 0 cell culture supernatant and a blank control were also included. The optimal working concentration was selected as the antigen dilution with an OD450 value around 1.0 and the highest P / N ratio. The results showed that the optimal antigen dilution was 1:1000 (final protein concentration of 1 μg / mL).

[0125] 2.2 Establishment of hybridoma cell lines containing anti-p30 protein monoclonal antibody (mAb) Using p30 protein as an immunogen, 6-8 week old female BALB / c mice were immunized according to standard methods. Spleen cells from immunized mice were then fused with well-developed SP2 / 0 myeloma cells using standard methods. Using p30 protein as the coating antigen, a newly established indirect ELISA method was employed to detect specific antibodies in the supernatant of the fused cells. Cells with high antibody titers, exhibiting monoclonal growth and good cell condition were selected for subclonal culture until antibody secretion stabilized and the proportion of positive wells exceeded 95%, at which point the culture was expanded.

[0126] 3. Preparation and potency determination of ascites fluid Hybridoma cells 2H12 were obtained through indirect ELISA screening. These cells were then cultured in a larger scale, and after routine treatment, ascites was prepared by intraperitoneal injection into Balb / c mice treated with incomplete Freund's adjuvant. The titers of the ascites were consistently 1:10 as determined by indirect ELISA. 6The above describes the purification of mAb in ascites fluid using the octanoic acid-ammonium sulfate method. Western blotting was used to determine the reactivity of the mAb (see [link to documentation]). Figure 2 Western blot results showed that 2H12 was reactive with prokaryotically expressed p30 protein, but not with the pET15b empty vector.

[0127] 4. Stability identification of monoclonal antibodies Indirect ELISA was used to determine the titer of the culture supernatant from the 10th, 20th, 30th, 40th, and 50th generations of screened 2H12 hybridoma cells to ascertain the stability of the hybridoma cells. The results showed that 2H12 hybridoma cells could stably secrete monoclonal antibodies, and the titers remained basically at the same level.

[0128] 5. Immunofluorescence identification of monoclonal antibodies SF9 cells were transfected with Bacmid-ASFV-p30 to package recombinant baculovirus. Hi5 cells were then infected with the P3 generation virus. Cells were seeded in 96-well plates according to cell number and cultured for 48 hours. The supernatant was discarded, and the cells were washed twice with PBS. Cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes. After washing three times with PBS, 100 μL of goat serum was added to each well, and the cells were blocked at 37°C for 1 hour. The blocking solution was discarded, and 100 μL of cell supernatant was added to each well for incubation. Mouse negative serum was used as a negative control, and the cells were incubated at 37°C for 1 hour. After washing three times with PBS, DAPI staining was performed, and the results were observed under a fluorescence microscope. Figure 3 As shown, green fluorescence was clearly observed in the cell supernatant of 2H12, while no green fluorescence was observed in the negative control, indicating that this 2H12 monoclonal antibody can specifically recognize the p30 protein.

[0129] 6. Identification of Monoclonal Antibody Subtypes The IgG subtype of the monoclonal antibody in ascites was identified using a subtype identification kit from Sigma-Aldrich, following the kit's instructions. Subtype identification revealed that the antibody subtype of 2H12 was IgG1 / k.

[0130] 7. Determination of monoclonal antibody sequence Once the hybridoma cells have grown into a monolayer, discard the culture medium, pipette and count the cells using 3 mL of sterile PBS, and collect 1×10⁶ cells. 6One cell was transferred to a 1.5 mL centrifuge tube and centrifuged at 800 rpm for 5 min. After discarding the supernatant, RNA was extracted from the hybridoma cells using the Trizol method. The VH and VL regions of the hybridoma cells were amplified using the Frdbio® mouse hybridoma cell monoclonal antibody variable region fragment amplification (sequencing) kit. The obtained target bands were recovered from the gel and cloned into the T vector. Positive clones were selected for sequencing, and the sequences of VH and VL were obtained. The amino acid / nucleotide sequences of the variable region and CDR of 2H12 hybridoma cells obtained by sequencing are shown in Table 1.

[0131] Example 2: Construction, Recombinant Expression and Functional Verification of Antibodies 1. Construction of recombinant plasmids for monoclonal antibodies The heavy chain (amino acid sequence shown in SEQ ID NO: 69) and light chain (amino acid sequence shown in SEQ ID NO: 70) of monoclonal antibody 2H12 were separately inserted into the pcDNA3.4 eukaryotic expression vector via gene synthesis to obtain recombinant expression plasmids of the heavy chain and light chain of monoclonal antibody 2H12, respectively (construction diagram of the recombinant expression plasmids is shown in...). Figure 4 , 5 The recombinant expression plasmids were transformed into DH10Bac competent cells and plated. Single colonies were picked and sequenced to identify positive colonies. The plasmids were then extracted for later use.

[0132] 2. Construction of bispecific antibody recombinant plasmid In this embodiment, a bispecific antibody was prepared using the monoclonal antibody 2H12, which specifically binds to the p30 protein of African swine fever virus, prepared in Example 1, and the monoclonal antibody MM1, which specifically binds to the E2 protein of classical swine fever virus, reported in the literature (Zhang J, Guo Z, Zhao Y, Yang Y, Huang P, Wang N, Qian Z, He M, Wu J, Luo L, Li Z and Pan C (2023), Identification of novel monoclonal antibodies specific for the conserved epitopes in the E2 protein of genotype 2 classical swine fever virus: implication for differential diagnosis. ActaVirol. 67:12124. doi: 10.3389 / av.2023.12124). The bispecific antibody in this embodiment possesses... Figure 6The structure shown connects the MM1 scFv antibody to the C-terminus of the 2H12 monoclonal antibody via a hinge structure, forming a bivalent bispecific antibody 2H12-MM1. This antibody can simultaneously bind to two antigens: ASFV p30 protein and CSFV E2 protein. In this embodiment, the amino acid sequence of the heavy chain of the bispecific antibody is shown in SEQ ID NO: 71, and the amino acid sequence of the light chain is shown in SEQ ID NO: 70.

[0133] The heavy chain of the bispecific antibody was synthesized and inserted into the pcDNA3.4 eukaryotic expression vector (Shanghai Newp Biotechnology Co., Ltd., catalog number V001453). The recombinant plasmid construction map of the bispecific antibody heavy chain is shown below. Figure 7 As shown. The construction of the recombinant plasmid for the light chain of the bispecific antibody is consistent with the construction of the recombinant plasmid for the 2H12 light chain of the monoclonal antibody, as shown. Figure 5 As shown in the figure. After transforming the above recombinant plasmid into DH10Bac competent cells, the cells were plated, and single colonies were picked, identified by sequencing, and the plasmid was extracted for later use.

[0134] 3. Antibody Expression and Purification 90 mL of HEK293F cells were adjusted to a density of 1.1 × 10⁻⁶. 6 100 μg of plasmid (50 μg heavy chain and 50 μg light chain plasmid, i.e., transient transfection with heavy chain and light chain plasmids at a 1:1 ratio) and 300 μL of PEI were added to 5 mL of Freestyle 293Expression medium, respectively. After thorough mixing, the mixture was incubated at room temperature for 5 min. The solution containing plasmid and PEI was then mixed and incubated at room temperature for 20 min. After incubation, the mixture was added to HEK293F cells, and the cell supernatant was collected after 5 days. The cell supernatant was filtered and purified using a protein A gel, eluted with 0.1 mol / L Glycine-HCl (pH 3.0), neutralized with 1 mol / L Tris-HCl (pH 9.0), concentrated using a 30 kDa ultrafiltration tube, and then replaced with phosphate buffer. The purified protein was subjected to reductive denaturation, and its size and purity were identified by SDS-PAGE. The heavy chain of monoclonal antibody 2H12 was approximately 50 kDa, and the light chain was approximately 24 kDa, consistent with the expected relative molecular mass. Figure 8 The heavy chain of the bispecific antibody is approximately 78 kDa, and the light chain is approximately 24 kDa, consistent with the expected relative molecular mass. Figure 9 Size exclusion chromatography was used to analyze the aggregation state and purity of monoclonal antibodies and bispecific antibodies. Neither monoclonal antibody 2H12 nor bispecific antibody 2H12-MM1 showed heavy or light chain byproducts, and no severe aggregation was observed. Figure 9 ).

[0135] 4. Verification of antibody-antigen binding properties 4.1 Construction of ASFV-p30 and CSFV-E2 recombinant plasmids The ASFV p30 gene sequence (585 bp between 124824 and 125408 in GenBank: MK333180.1) was codon-optimized and inserted between EcoR I and Xho I in the pCAGGS vector (with 10 His molecules introduced at the N-terminus) to obtain the recombinant plasmid pCAGGS-p30. This plasmid was transformed into E. coli DH5α competent cells, plated, and cultured overnight. The next day, positive clones were picked and sequenced. The correctly sequenced positive bacterial cultures were extracted to obtain the correctly sequenced recombinant plasmid pCAGGS-p30 for later use.

[0136] The classical swine fever virus E2 gene sequence (GenBank: KT953611.1) was inserted into the EcoRI and XhoI space of the pCAGGS vector after codon optimization using the same method described above. After transformation and identification, the recombinant plasmid pCAGGS-E2 was obtained for later use.

[0137] 4.2 Verification of antibody-antigen binding properties HEK293T cells were transiently transfected with the recombinant plasmids pCAGGS-p30 and pCAGGS-E2 using a Lipo3000 transfector to construct HEK293T-p30 and HEK293T-E2 cells, respectively. Wild-type HEK293T cells and HEK293T-p30 cells were digested and incubated with different concentrations of the monoclonal antibody 2H12. After washing twice with PBS, the cells were stained with APC-goat anti-mouse IgG antibody (Biolegend, catalog number 405308). After washing twice with PBS, the cell fluorescence intensity was detected by flow cytometry. Wild-type HEK293T cells, HEK293T-p30 cells, and HEK293T-E2 cells were digested and incubated with different concentrations of the bispecific antibody 2H12-MM1. After washing twice with PBS, the cells were stained with APC-goat anti-mouse IgG antibody (Biolegend, catalog number 405308). After washing twice with PBS, the cell fluorescence intensity was detected by flow cytometry.

[0138] The results are as follows Figure 10 As shown: monoclonal antibody 2H12 can bind to HEK293T-p30 cells, but its binding to wild-type HEK293T cells is very weak. Figure 10 (A). The bispecific antibody 2H12-MM1 can bind to both HEK293T-p30 and HEK293T-E2 cells, but its binding to wild-type HEK293T cells is very weak. This indicates that the preparation of the bispecific antibody did not change the antigen-binding characteristics of antibody 2H12 and antibody MM1. Figure 10(B)

[0139] 5. Identification of antibody specificity The specificity of monoclonal antibody 2H12 and bispecific antibody 2H12-MM1 was identified using ELISA. The specific method is as follows: The target antigen p30 protein (prepared in Example 1), CSFV vaccine virus (Wuhan Keqian Biotechnology Co., Ltd.), and unrelated viral vaccine viruses—porcine circovirus type 2 inactivated vaccine (LG strain) (Harbin Veterinary Research Institute), porcine reproductive and respiratory syndrome live vaccine (Boehringer Ingelheim), and porcine pseudorabies live vaccine (Kartha-K61 strain) (Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.)—were dissolved in coating buffer and coated onto 96-well ELISA plates (NC indicates coated BSA), 200 ng / 100 μL per well, incubated overnight at 4°C; the buffer was discarded, washed twice with PBS, and blocked for 1 h with PBS containing 5% skim milk powder at 200 μL / well; the buffer was discarded, and the test antibody (diluted with PBS containing 5% skim milk powder and 0.1% Tween-20, hereinafter the same) was added at 50 μL / well, and reacted at 37°C for 1 h. h; Discard the solution, wash three times with PBS containing 0.1% Tween-20, add 50 μL / well of HRP-labeled goat anti-mouse IgG (Zhongshan Jinqiao Biotechnology Co., Ltd.) (1:2000 dilution), and react at 37℃ for 45 min; Discard the solution; wash three times with PBS containing 0.1% Tween-20; add 100 μL / well of freshly prepared TMB chromogenic solution, and react at room temperature for 15 min; terminate the chromogenic reaction by adding 50 μL / well of 2 mol / L concentrated sulfuric acid, and measure the absorbance value at 450 nm; perform one-way ANOVA to analyze the differences in the results. The results are as follows. Figure 11 As shown: Monoclonal antibody 2H12 exhibits strong binding activity to the p30 protein, but shows almost no binding with irrelevant viruses CSFV, PCV2, PRRSV, PRV, and the BSA negative control, indicating that the 2H12 antibody prepared in this invention has good specificity. Figure 11 (A). The bispecific antibody 2H12-MM1 exhibits strong binding activity against both p30 protein and CSFV vaccine, but shows almost no binding with unrelated viruses PCV2, PRRSV, PRV, and the BSA negative control, indicating that the 2H12-MM1 bispecific antibody prepared in this invention still possesses good specificity, and the realization of the dual epitope recognition function has not altered its specificity. Figure 11 (B)

[0140] Example 3: Preparation and application of colloidal gold test strips 1. Preparation of colloidal gold solution Dilute a 1% chloroauric acid solution to 0.01% (mass fraction) with double-distilled deionized water. Place 100 mL of the 0.01% chloroauric acid solution in an Erlenmeyer flask and heat to boiling using a constant-temperature electromagnetic stirrer. Quickly add 1.6 mL of 1% trisodium citrate solution at once and continue stirring at a constant speed. Stop stirring when the solution turns a clear red color. After the color stabilizes, continue heating for 5 minutes. After cooling to room temperature, restore the original volume with deionized water to obtain a colloidal gold solution, which should be stored at 4℃.

[0141] 2. Preparation of bispecific antibody 2H12-MM1-colloidal gold label Take 100 mL of the prepared 0.01% colloidal gold solution, and adjust the pH of the colloidal gold solution to about 8.5 with 0.1 mol / L potassium carbonate under magnetic stirring. Add the bispecific antibody 2H12-MM1 prepared in Example 2 to the colloidal gold solution according to the standard of adding 20-40 μg of bispecific antibody per mL of colloidal gold solution. After stirring evenly, let it stand at room temperature (25°C) for 20 min. Add 1 mL of 10% bovine serum albumin (BSA) solution and stir with a magnetic stirrer (150 rpm for 30 min). After standing for 20 min, centrifuge at 12000 rpm for 40 min at 4 °C. Discard the supernatant and resuspend the remaining precipitate in 20 mL of colloidal gold resuspension buffer (2% sucrose, 1% BSA, 0.3% Tween 20, prepared with 0.01 mol / L phosphate buffer solution at pH 7.4) to prepare the bispecific antibody 2H12-MM1-colloidal gold label, which is stored at 4 °C for later use.

[0142] 3. Preparation of ASFV-CSFV colloidal gold test strips The bispecific antibody 2H12-MM1-colloidal gold label was uniformly sprayed onto the gold-labeled pad at a rate of 5 μL / cm using a spray membrane apparatus. The pad was dried at 37°C for 2 hours. The NC membrane has a detection zone (T line) and a control zone (C line). The detection zone (T line) is coated with the bispecific antibody 2H12-MM1 prepared in Example 2 of this invention at a concentration of 0.2–0.8 mg / mL. The control zone (C line) is coated with goat anti-mouse monoclonal antibody (Merck, catalog number M4280) at a concentration of 0.2–0.8 mg / mL. The NC membrane, gold-labeled pad, sample pad, and absorbent pad were sequentially attached to a PVC base plate. After attachment, the strips were cut into 4 mm wide test strips, placed in a specially made test strip plastic card, and then placed in a sealed bag with desiccant. The bag was sealed to obtain a colloidal gold chromatography test strip capable of simultaneously detecting African swine fever virus and classical swine fever virus. The strips were stored at room temperature.

[0143] 4. Application of colloidal gold test strips 4.1 Sample preparation: Whole blood samples can be tested directly or diluted appropriately with diluent before testing; tissue samples are homogenized by adding an appropriate amount of PBS, centrifuged at 2000 rpm to remove tissue sediment, and the supernatant is used directly for testing.

[0144] 4.2 Detection: Add 3-5 drops of the sample to be tested into the sample well of the colloidal gold chromatography test strip. After 5-10 minutes, interpret the results.

[0145] 4.3 Result Judgment: The results are determined by comparing the color intensity of the quality control area (C line) and the detection area (T line).

[0146] Judgment criteria: Positive: When both the control area (C line) and the test area (T line) show bands, it is judged as positive, that is, the sample to be tested contains ASFV or CSFV or both ASFV and CSFV, and is indicated by "+"; Negative: When the control area (C line) shows a band and the detection area (T line) does not show color, it is judged as negative, that is, the sample to be tested does not contain ASFV and CSFV or the content of the two viruses is lower than the detection limit, which is indicated by "-"; Invalid: If no band is displayed in the control area (C line), the test strip is invalid.

[0147] The principle for determining the above results is as follows: Figure 12 .

[0148] 4.4 Sample Testing: Forty clinically collected pig blood samples were tested using the colloidal gold chromatography test strip prepared in this embodiment. Positive blood samples were further serially diluted and simultaneously tested with serially diluted CSFV vaccine virus to test the sensitivity of the test strip. Control viruses (PRRSV vaccine virus, PCV2 vaccine virus, PRV vaccine virus, and pig negative serum) were tested to test the specificity of the test strip. The test results are shown in Table 2.

[0149] Table 2: Measured Results of Colloidal Gold Test Strips

[0150] As shown in Table 2, the colloidal gold chromatography detection card prepared in this embodiment can effectively detect ASFV or CSFV positive blood samples in field samples. The sensitivity for detecting clinical positive blood samples can be as low as 1:300 dilution, and for CSFV vaccine, it can be detected at 1:500 dilution. It has high sensitivity and good specificity, and there is no cross-reactivity with PRRSV vaccine virus, PCV2 vaccine virus, PRV vaccine virus, and porcine negative serum.

[0151] 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 p30 protein of African swine fever virus, wherein the antibody or antigen-binding fragment thereof comprises: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; Wherein, the CDR is defined according to the IMGT numbering system; or The antibody or its antigen-binding fragment includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; Wherein, the CDR is defined according to the Kabat numbering system; or The antibody or its antigen-binding fragment includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 30, HCDR2 having the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; Wherein, the CDR is defined according to the Chothia numbering system; or The antibody or its antigen-binding fragment includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 32, HCDR2 having the amino acid sequence shown in SEQ ID NO: 33, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 34; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 35, LCDR2 having the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 37; The CDR is defined according to the Contact numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment further includes a framework region of the heavy chain variable region; and / or the light chain variable region of the antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment includes: The heavy chain variable region (VH) includes the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region (VL) includes the amino acid sequence shown in SEQ ID NO:

18.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

5. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 66; the light chain constant region includes the amino acid sequence shown in SEQ ID NO:

68.

6. A bispecific antibody comprising two protein functional regions targeting antigens, wherein one protein functional region comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5.

7. The bispecific antibody according to claim 6, characterized in that, The bispecific antibody includes a first protein functional region that specifically binds to the African swine fever virus p30 protein and a second protein functional region that specifically binds to the swine fever virus E2 protein. The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 42, HCDR2 having the amino acid sequence shown in SEQ ID NO: 43, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 44; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 45, LCDR2 having the amino acid sequence shown in SEQ ID NO: 46, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; The CDR is defined according to the IMGT numbering system; or, The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 48, HCDR2 having the amino acid sequence shown in SEQ ID NO: 49, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 50; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 51, LCDR2 having the amino acid sequence shown in SEQ ID NO: 52, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; The CDR is defined according to the Kabat numbering system; or, The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 30, HCDR2 having the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 28, LCDR2 having the amino acid sequence shown in SEQ ID NO: 29, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 53, HCDR2 having the amino acid sequence shown in SEQ ID NO: 54, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 50; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 51, LCDR2 having the amino acid sequence shown in SEQ ID NO: 52, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; The CDR is defined according to the Chothia numbering system; or, The first protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 32, HCDR2 having the amino acid sequence shown in SEQ ID NO: 33, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 34; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 35, LCDR2 having the amino acid sequence shown in SEQ ID NO: 36, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 37; The second protein functional region includes: VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 55, HCDR2 having the amino acid sequence shown in SEQ ID NO: 56, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 57; and VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 58, LCDR2 having the amino acid sequence shown in SEQ ID NO: 59, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 60; The CDR is defined according to the Contact numbering system.

8. The bispecific antibody according to claim 7, characterized in that, The heavy chain variable region of the first protein functional region also includes the framework region of the heavy chain variable region; and / or The heavy chain variable region of the second protein functional region also includes the framework region of the heavy chain variable region; and / or The light chain variable region of the first protein functional region also includes the framework region of the light chain variable region; and / or The light chain variable region of the second protein functional region also includes the framework region of the light chain variable region.

9. The bispecific antibody according to claim 7, characterized in that, The first protein functional region includes: The heavy chain variable region (VH) includes the amino acid sequence shown in SEQ ID NO: 17; and the light chain variable region (VL) includes the amino acid sequence shown in SEQ ID NO: 18; The second protein functional region includes: The heavy chain variable region (VH) includes the amino acid sequence shown in SEQ ID NO: 40; and the light chain variable region (VL) includes the amino acid sequence shown in SEQ ID NO:

41.

10. The bispecific antibody according to claim 7, characterized in that, The first and second protein functional regions are directly connected or connected through a linker fragment.

11. The bispecific antibody according to claim 7, characterized in that, The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain Fv.

12. The bispecific antibody according to claim 11, characterized in that, The single-chain Fv is attached to the C-terminus of the heavy chain of the immunoglobulin via a linker fragment.

13. The bispecific antibody according to claim 12, characterized in that, The bispecific antibody comprises a first polypeptide chain and a second polypeptide chain: The first polypeptide chain comprises: the heavy chain of the immunoglobulin, the linker fragment, and the single-chain Fv; the second polypeptide chain comprises: the light chain of the immunoglobulin.

14. The bispecific antibody according to claim 13, characterized in that, The first polypeptide chain comprises amino acids 19-741 of SEQ ID NO: 71; and The second polypeptide chain includes the amino acid sequence from position 19 to 231 of SEQ ID NO:

70.

15. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 5, or a nucleotide sequence encoding a bispecific antibody as described in any one of claims 6 to 14.

16. A carrier comprising the nucleic acid molecule of claim 15.

17. A host cell comprising the nucleic acid molecule of claim 15 or the vector of claim 16.

18. A conjugate comprising the antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, or the bispecific antibody as claimed in any one of claims 6 to 14; and, a conjugation portion.

19. A detection product comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, or a bispecific antibody as described in any one of claims 6 to 14, a nucleic acid molecule as described in claim 15, or a vector as described in claim 16, or a host cell as described in claim 17, or a conjugate as described in claim 18.

20. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in any one of c1)-c6): c1) Prepare products for the diagnosis of African swine fever virus infection or the disease it causes; c2) Prepare products for the prevention and / or treatment of African swine fever virus infection or diseases caused by it; c3) Prepare products for detecting the presence or level of African swine fever virus or its p30 protein in samples; c4) Detect the presence or level of African swine fever virus or its p30 protein in the sample; c5) Screening for drugs to prevent and / or treat African swine fever virus infection or the disease it causes; c6) Products for the preparation of drugs for screening the prevention and / or treatment of African swine fever virus infection or diseases caused by it.

21. The use of the bispecific antibody according to any one of claims 6 to 14 in any one of c1)-c6): c1) Prepare products for the diagnosis of African swine fever virus and / or classical swine fever virus infection or diseases caused by it; c2) Prepare products for the prevention and / or treatment of African swine fever virus and / or classical swine fever virus infection or diseases caused by it; c3) Prepare products for detecting the presence or level of African swine fever virus, African swine fever virus p30 protein, classical swine fever virus and / or classical swine fever virus E2 protein in samples; c4) Detect the presence or level of African swine fever virus, African swine fever virus p30 protein, classical swine fever virus and / or classical swine fever virus E2 protein in the sample; c5) Screening for drugs to prevent and / or treat African swine fever virus and / or classical swine fever virus infection or the disease it causes; c6) Products for the preparation of drugs for screening the prevention and / or treatment of African swine fever virus and / or classical swine fever virus infection or diseases caused by it.

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