CN121517546A - Monoclonal antibodies that specifically bind to the African swine fever virus EP153R protein and their applications - Google Patents

Abstract

The invention provides a monoclonal antibody specifically combined with an African swine fever virus EP153R protein and application thereof, wherein the antibody has high titer and stable property, can be combined with ASFV infected cells, and provides an important tool for ASFV detection, EP153R protein detection and EP153R protein structure analysis.

Description

Monoclonal antibody specifically binding with African swine fever virus EP153R protein and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a monoclonal antibody specifically combined with an African swine fever virus EP153R protein and application thereof.

Background

African swine fever (AFRICAN SWINE FEVER, ASF) is an acute, virulent, highly contagious infectious disease of pigs caused by infection of domestic pigs and wild pigs with African Swine Fever Virus (ASFV), and is characterized by high fever, poor appetite, cyanosis of skin and bleeding of internal organs, with short course of disease and mortality up to 100%. ASF is a legal report of animal epidemic disease by the world animal health Organization (OIE), a prescribed class of animal epidemic disease in our country, and is highly valued worldwide. The disease is first confirmed to occur in kennia in the africa in 1921, and africa swine fever occurs, spreads and is popular in a plurality of countries worldwide in 2007, and the disease is transmitted into China in 2018, so that huge direct and indirect economic losses are brought about. The disease is found to be almost hundreds of years at home and abroad, and a great deal of research work is done on ASF by students at home and abroad, but research shows that the inactivated vaccine has an unobvious effect, the attenuated vaccine has a certain protection effect but has poor safety, and at present, no vaccine for effectively preventing ASF and no specific medicine for treating the disease exist globally. Control of ASF currently relies on rapid diagnosis, the killing of diseased animals, and the adoption of effective quarantine and strict sanitation.

ASFV is the only member of the African swine fever related virus family (ASFARVIRIDAE), african swine fever virus genus (Asfivirus), and is also the only known DNA arbovirus. ASFV is a large and complex icosahedral double stranded linear DNA virus (170-194 kb) with double-layered envelope, the virion diameter is about 260nm, contains 151-167 open reading frames, and encodes 150-200 proteins. ASFV mainly infects monocytes and alveolar macrophages, and viral invasion is completed through endocytosis, megaloblastic and other actions, and DNA replication, viral assembly and release are all carried out in macrophages.

The EP153R gene is located in EcoRI E' fragment of ASFV genome, its full length is 474bp, and its code can produce lectin-like membrane protein containing 158 amino acids, and is one of several glycosylated membrane proteins of ASFV. The protein comprises an N-glycosylation site, a phosphorylation site, an acylation site, a transmembrane region, a C-type animal lectin domain and a cell attachment sequence, and is expressed in the early and late stages of virus infection. EP153R, also known as C-type lectin, is one of the factors related to viral virulence and plays a key role in the mechanism of infection, immune escape and vaccine research of viruses. Comparative genomics shows that the locus containing adjacent CD2v and C-type lectin genes is one of the most variable regions in the ASFV genome, and that CD2v and EP153R proteins together mediate the erythrocyte adsorption phenomenon of ASFV. In addition, research shows that the attenuated strain can effectively resist the attack of homologous and strong strains, but when EP153R and CD2v of the attenuated strain are replaced by homologous genes of heterologous strains, the protection effect of the attenuated strain on the original homologous strain is completely lost, which indicates that the CD2v and the EP153R are important protective antigens of ASFV. Notably, the NH/P68 strain with the EP153R gene knocked out produced 100% immune protection against homologous ASFV challenge. In summary, EP153R is a core candidate protein for ASFV subunit vaccine development. The preparation of the antibody capable of specifically binding to the EP153R protein is not only beneficial to deeply analyzing the biological function of the protein and establishing an accurate protein detection method, but also can provide key technical support for the research and development of African swine fever vaccines.

Disclosure of Invention

In order to solve one of the technical problems in the prior art, the invention provides a monoclonal antibody specifically binding with an African swine fever virus EP153R protein and application thereof.

In a first aspect the present invention provides an antibody or antigen binding fragment thereof which specifically binds to the EP153R protein of african swine fever virus, wherein the antibody or antigen binding fragment thereof comprises Complementarity Determining Regions (CDRs) as follows:

(a1) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region shown in SEQ ID NO. 32 and/or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region shown in SEQ ID NO. 33;

(a2) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region, and/or CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region described below, wherein at least one CDR contains a mutation that is a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared to the heavy chain variable region and/or light chain variable region described in (a 1).

In some embodiments, the substitution is a conservative substitution.

In some embodiments, the complementarity determining regions are defined according to Kabat, chothia, IMGT, contact, or AbM numbering system.

In some embodiments, the antibody or antigen binding fragment thereof comprises the heavy chain variable region and/or light chain variable region, wherein the complementarity determining regions are defined according to the IMGT numbering system:

A heavy chain variable region comprising 3 CDRs of CDR-H1 of SEQ ID NO. 36 or a variant thereof, CDR-H2 of SEQ ID NO. 37 or a variant thereof, CDR-H3 of SEQ ID NO. 38 or a variant thereof, and/or a light chain variable region comprising 3 CDRs of CDR-L1 of SEQ ID NO. 48 or a variant thereof, CDR-L2 of SEQ ID NO. 49 or a variant thereof, CDR-L3 of SEQ ID NO. 50 or a variant thereof;

wherein the variant has a substitution, deletion, or addition of one or more amino acids compared to the sequence from which it is derived, and in some embodiments the substitution is a conservative substitution (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids).

In some embodiments, the antibody or antigen binding fragment thereof comprises the heavy chain variable region and/or the light chain variable region, wherein the complementarity determining regions are defined according to the Kabat numbering system:

A heavy chain variable region comprising 3 CDRs of CDR-H1 of SEQ ID NO 39 or a variant thereof, CDR-H2 of SEQ ID NO 40 or a variant thereof, CDR-H3 of SEQ ID NO 41 or a variant thereof, and/or a light chain variable region comprising 3 CDRs of CDR-L1 of SEQ ID NO 51 or a variant thereof, CDR-L2 of SEQ ID NO 52 or a variant thereof, CDR-L3 of SEQ ID NO 53 or a variant thereof;

wherein the variant has a substitution, deletion, or addition of one or more amino acids compared to the sequence from which it is derived, and in some embodiments the substitution is a conservative substitution (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids).

In some embodiments, the antibody or antigen binding fragment thereof comprises the heavy chain variable region and/or light chain variable region, wherein the complementarity determining regions are defined according to the Chothia numbering system:

a heavy chain variable region comprising 3 CDRs of CDR-H1 of SEQ ID NO. 42 or a variant thereof, CDR-H2 of SEQ ID NO. 43 or a variant thereof, CDR-H3 of SEQ ID NO. 44 or a variant thereof, and/or a light chain variable region comprising 3 CDRs of CDR-L1 of SEQ ID NO. 51 or a variant thereof, CDR-L2 of SEQ ID NO. 52 or a variant thereof, CDR-L3 of SEQ ID NO. 53 or a variant thereof;

wherein the variant has a substitution, deletion, or addition of one or more amino acids compared to the sequence from which it is derived, and in some embodiments the substitution is a conservative substitution (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids).

In some embodiments, the antibody or antigen binding fragment thereof comprises the heavy chain variable region and/or the light chain variable region, wherein the complementarity determining regions are defined according to the Contact numbering system:

A heavy chain variable region comprising 3 CDRs of CDR-H1 of SEQ ID NO 45 or a variant thereof, CDR-H2 of SEQ ID NO 46 or a variant thereof, CDR-H3 of SEQ ID NO 47 or a variant thereof, and/or a light chain variable region comprising 3 CDRs of CDR-L1 of SEQ ID NO 54 or a variant thereof, CDR-L2 of SEQ ID NO 55 or a variant thereof, CDR-L3 of SEQ ID NO 56 or a variant thereof;

wherein the variant has a substitution, deletion, or addition of one or more amino acids compared to the sequence from which it is derived, and in some embodiments the substitution is a conservative substitution (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids).

In some embodiments, the antibody or antigen binding fragment thereof comprises:

A heavy chain variable region comprising a sequence as set forth in SEQ ID NO. 32 or a variant thereof and/or a light chain variable region comprising a sequence as set forth in SEQ ID NO. 33 or a variant thereof;

Wherein 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 to the sequence from which it is derived, or has a substitution, deletion, or addition of one or more amino acids to the sequence from which it is derived, and in some embodiments, the substitution is a conservative substitution.

In some embodiments, the antibody or antigen binding fragment thereof is a murine antibody, a chimeric antibody, or a porcine antibody.

In some embodiments, the antibody is of IgA, igD, igE, igG or IgM type.

In some embodiments, the antibodies are of the IgG1, igG2, igG3, igG4, igA1 and IgA2 types.

In some embodiments, the antibody or antigen binding fragment thereof further comprises a constant region derived or derived from a murine immunoglobulin, or a constant region derived or derived from a porcine immunoglobulin.

In some embodiments, the heavy chain of the antibody or antigen binding fragment thereof comprises a heavy chain constant region derived or derived from a murine immunoglobulin.

In some embodiments, the antibody or antigen binding fragment thereof comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region having altered effector function as compared to the wild-type Fc region.

In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region derived or derived from murine IgG2 a.

In some embodiments, the light chain of the antibody or antigen binding fragment thereof comprises a light chain constant region derived or derived from a murine immunoglobulin.

In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain constant region derived or derived from murine igkappa.

In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO. 57 or a variant thereof having a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 15, up to 10, or up to 5 amino acids; e.g., a conservative substitution of 1,2, 3, 4, or 5 amino acids) as compared to SEQ ID NO. 57.

In some embodiments, the antibody or antigen binding fragment thereof comprises a light chain constant region as shown in SEQ ID NO. 58 or a variant thereof having a conservative substitution of up to 20 amino acids (e.g., a conservative substitution of up to 15, up to 10, or up to 5 amino acids; e.g., a conservative substitution of 1,2, 3, 4, or 5 amino acids) as compared to SEQ ID NO. 58.

In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain constant region as set forth in SEQ ID NO. 57 and a light chain constant region as set forth in SEQ ID NO. 58.

In some embodiments, the antibody or antigen binding fragment thereof comprises:

A heavy chain comprising a heavy chain variable region as set forth in SEQ ID NO. 32 and a heavy chain constant region as set forth in SEQ ID NO. 57, and a light chain comprising a light chain variable region as set forth in SEQ ID NO. 33 and a light chain constant region as set forth in SEQ ID NO. 58.

In some embodiments, the antibody or antigen binding fragment thereof is ScFv, fab, fab ', fab ' -SH, F (ab ') 2, fv fragment, disulfide-linked Fv (dsFv), diabody (diabody), bispecific antibody, or multispecific antibody.

In some embodiments, the antibody or antigen binding fragment thereof is labeled.

In some embodiments, the antibody or antigen binding fragment thereof carries a detectable label, which may be any substance that is detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, chemical, and the like. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-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 incorporation of the above-described marker-modified avidin (e.g., streptavidin). In some embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label is selected from the group consisting of a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. In some embodiments, the detectable label as described above may be attached to an antibody or antigen binding fragment thereof of the disclosure that specifically binds to the african swine fever virus EP153R protein via a linker (linker) of different length to reduce potential steric hindrance.

In a second aspect the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof according to the first aspect, a heavy and/or light chain thereof, a heavy chain variable region thereof and/or a light chain variable region thereof. In certain embodiments, the nucleotide sequence is replaceable according to codon degeneracy in the art. In certain embodiments, the nucleotide sequence is codon optimized.

In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, the nucleic acid molecule encoding an antibody heavy chain variable region comprising (i) a nucleotide sequence as set forth in SEQ ID NO: 34, (ii) a sequence substantially identical to SEQ ID NO: 34 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity as compared to SEQ ID NO: 34, or a sequence having one or more nucleotide substitutions), or (iii) a degenerate sequence as set forth in (i) or (ii) above, and/or (iv) a sequence substantially identical to SEQ ID NO: 35 (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more sequence identity as compared to SEQ ID NO: 35, or a sequence having one or more nucleotide substitutions), or (iv) a degenerate sequence as set forth in (vi) or (v).

In a third aspect the invention provides a vector comprising a nucleic acid molecule according to the second aspect.

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

In some embodiments, the vector is an expression vector. In some embodiments, the expression vector may include a eukaryotic expression vector and/or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vectors include, for example, but are 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, a plasmid, a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

In some embodiments, the carrier may be selected from nanoparticles, liposomes, exosomes, microbubbles, or gene-guns.

In a fourth aspect the invention provides a host cell comprising a nucleic acid molecule according to the second aspect or a vector according to the third aspect.

In some embodiments, the host cell is not involved in propagation material.

In some embodiments, the host cell may be a host cell conventionally used in the art, provided that the expression vector is capable of stably expressing the carried nucleic acid molecule as an antibody or antigen-binding fragment thereof, chimeric antigen receptor or multispecific antibody or antigen-binding fragment thereof of the disclosure that specifically binds the african swine fever virus EP153R protein described above. In some embodiments, the host cell may be a prokaryotic cell and/or a eukaryotic cell, which may include, for example, E.coli, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NS0 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.

In a fifth aspect the invention provides a method of preparing an antibody or antigen-binding fragment thereof according to the first aspect, comprising culturing the host cell according to the fourth aspect 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.

In a sixth aspect the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect, or a nucleic acid molecule according to the second aspect, or a vector according to the third aspect, or a host cell according to the fourth aspect, and a pharmaceutically acceptable carrier and/or excipient.

In a seventh aspect the invention provides a test kit comprising an antibody or antigen binding fragment thereof according to the first aspect, or a nucleic acid molecule according to the second aspect, or a vector according to the third aspect, or a host cell according to the fourth aspect, for use in detecting the presence or level of african swine fever virus or african swine fever virus EP153R protein in a sample.

According to an eighth aspect of the invention there is provided the use of an antibody or antigen binding fragment thereof according to the first aspect, or a nucleic acid molecule according to the second aspect, or a vector according to the third aspect, a host cell according to the fourth aspect, or a pharmaceutical composition according to the sixth aspect, in the preparation of a reagent for one or more of the following uses:

1) Detecting the presence or level of african swine fever virus or african swine fever virus EP153R protein in the sample;

2) Preventing and/or treating diseases related to African swine fever virus.

In some embodiments, the EP153R protein has the amino acid sequence shown in SEQ ID NO. 1 or a variant thereof 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 to the sequence from which it is derived, or has a substitution, deletion, or addition of one or several amino acids compared to the sequence from which it is derived, in some embodiments, the substitution is a conservative substitution (e.g., a substitution, deletion, or addition of 1,2, or 3 amino acids).

In some embodiments, the african swine fever virus-related disease is african swine fever.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides a monoclonal antibody specifically combined with an African swine fever virus EP153R protein, which has high titer and stable property, can be combined with African swine fever virus infected cells, and can be used for ELISA, flow cytometry, western blot and immunohistochemical detection. The variable region sequence of the monoclonal antibody provided by the invention can be used for constructing complete genetic engineering antibodies and can also be used for expressing Fab antibodies, thereby providing an important tool for African swine fever virus detection, EP153R protein detection and EP153R protein structural analysis.

Drawings

FIG. 1 shows a SDS-PAGE quantitative view of EP153R recombinant proteins.

Figure 2 shows the binding of EP153R recombinant protein to african swine fever positive serum.

FIG. 3 shows the results of ELISA assays of hybridoma cell HY002 culture supernatant with EP153R protein, wherein EP 153R-immunized positive mouse serum was used as a positive control and non-immunized mouse serum was used as a negative control.

FIG. 4 shows the result of SDS-PAGE electrophoresis of purified monoclonal antibody HY 002.

FIG. 5 shows the identification results of HY002 monoclonal antibody subtype.

FIG. 6 shows the results of flow cytometry detection of extracellular portion of HY002 binding to EP153R protein.

FIG. 7 shows the result of western blot detection of HY002 binding specifically to EP153R protein.

FIG. 8 shows the binding of HY002 to ASFV virus infected cells, where A is the result of immunohistochemical staining and B is the result of Western blot detection.

FIG. 9 shows quality and binding titer detection of genetically engineered antibody enHY, wherein A is SDS-PAGE electrophoresis identification result and B is titer detection result.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

Abbreviations

IMGT is based on the numbering system of the international immunogenetics information system (The international ImMunoGeneTics information system (IMGT)) initiated by Lefranc et al, see LEFRANC ET al, dev. Comparat. Immunol. 27:55-77, 2003.

Kabat: immunoglobulin alignment and numbering System as proposed by Elvin A. Kabat (see, e.g.) Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

Chothia, an immunoglobulin numbering system proposed by Chothia et al, is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, e.g., chothia & Lesk (1987) J. Mol. Biol. 196:901-917; chothiaet al (1989) Nature 342:878-883).

AbM CDR definition mode, derived from Martin related research (Martin AC, Cheetham JC, Rees AR(1989). Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272).

Contact CDR definition mode, related research from Martin (Martin, A.C.R.(2001). Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Lab Manuals. Springer, Berlin, Heidelberg.).

ELISA, enzyme-linked immunosorbent assay.

PCR, polymerase chain reaction.

HRP horseradish peroxidase.

CDR-H1 complementarity determining region 1 in the heavy chain variable region of an immunoglobulin.

CDR-H2 complementarity determining region 2 in the immunoglobulin heavy chain variable region.

CDR-H3 complementarity determining region 3 in the immunoglobulin heavy chain variable region.

CDR-L1 complementarity determining region 1 in the immunoglobulin light chain variable region.

CDR-L2 complementarity determining region 2 in the immunoglobulin light chain variable region.

CDR-L3 complementarity determining region 3 in the immunoglobulin light chain variable region.

Definition of the definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

The term "antibody" as used herein refers to an immunoglobulin produced by the immune system upon stimulation of an antigen by proliferation and differentiation of B lymphocytes into plasma cells, which specifically binds to the corresponding antigen and mediates immune effects, mainly in serum and body fluids, and is an important immune molecule mediating humoral immunity. Antibodies can encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, or tetraspecific antibodies), single chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulins (Ig).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprised by the population are identical and/or bind to the same epitope, except for possibly minor amounts of variant antibodies (e.g., comprising naturally occurring mutations or produced during production of a monoclonal antibody preparation, typically in minor amounts). Unlike polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.

The term "multispecific antibody" as used herein is used in its broadest sense to encompass antibodies having multiple epitope specificities. Such 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 units have polyepitopic 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 linked together covalently or non-covalently, and the like.

The terms "full length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody that is substantially similar in structure to a natural antibody. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG class antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two disulfide-bonded light chains and two heavy chains. From N-terminal to C-terminal, each heavy chain has a variable region (VH) (also known as a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH 1, CH2, and CH 3) (also known as heavy chain constant regions, CH). From N-terminal to C-terminal, each light chain has a variable region (VL) (also known as a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also known as a light chain constant region). The heavy chain of an antibody may be one of five types, α (IgA), δ (IgD), epsilon (IgE), γ (IgG), or μ (IgM), and may be further divided into subtypes such as γ1 (IgG 1), γ2 (IgG 2), γ3 (IgG 3), γ4 (IgG 4), α1 (IgA 1), and α2 (IgA 2). The light chain of an antibody may be one of two types, a kappa (kappa) light chain and a lambda (lambda) light chain, based on the amino acid sequence of its constant domain.

Within the light and heavy chains, the variable and constant regions are linked by a "J" region containing about 12 or more amino acid residues, and the heavy chain also comprises a "D" region of about 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 3 domains (CH 1, CH2 and CH 3). 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 may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "Fd fragment" as used herein means an antibody fragment consisting of VH and CH1 domains. The term "dAb fragment" as used herein means an antibody fragment consisting of a VH domain (Wardet al., nature 341:544-546 (1989)). The term "Fab fragment" as used herein means an antibody fragment consisting of VL, VH, CL and CH1 domains. The term "F (ab') 2 fragment" as used herein means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. The term "Fab 'fragment" as used herein means the fragment obtained after reduction of the disulfide bond joining two heavy chain fragments in a F (ab') 2 fragment, consisting of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains). The term "Fab' -SH" as used herein refers to Fab fragments containing free sulfhydryl groups.

The term "Fv fragment" as used herein means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

The term "scFv" as used herein refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker. In some cases, disulfide bonds may also exist between VH and VL of scFv.

The term "variable region" or "variable domain" as used herein refers to the domain of an antibody heavy or light chain that is involved in binding an antigen binding molecule to an antigen. The variable regions of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FR 1-4) and three hypervariable regions (HVR 1-3), arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain may be sufficient to confer antigen binding specificity. Together, the 3 HVRs within VH and VL constitute the antigen binding site of Ig, which is capable of complementary binding to the corresponding epitope, so HVRs are also known as complementarity determining regions (complementarity determing region, CDRs) denoted by CDR1, CDR2 and CDR3, respectively. The VH or VL chain of an antibody may further comprise all or part of the heavy or light chain constant region.

CDRs of an antibody or antigen binding fragment thereof of the present disclosure can be determined according to various numbering systems known in the art. In some embodiments, the CDRs contained in an antibody or antigen binding fragment thereof of the present disclosure are preferably determined by the IMGT, kabat, contact, chothia or AbM numbering system.

The term "variable" as used herein refers to certain segments of the variable region that differ in sequence throughout antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the variable regions, but is concentrated in three segments within the light and heavy chain variable regions, known as hypervariable regions (HVRs). The relatively highly conserved portions of the variable regions are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three HVRs, which form loop junctions and in some cases form part of a β -sheet structure. The HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antigen binding site of the antibody. The constant region is not directly involved in binding of the antibody to the antigen, and has other effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain of an antibody has. The heavy chain structure and antigenicity can be classified into 5 classes (class) consisting of mu chain, gamma chain, alpha chain, delta chain and epsilon chain, and immunoglobulins consisting of different heavy and light chains are called IgA, igD, igE, igG and IgM, respectively. The same class of Ig has different amino acid composition in hinge region and different number and position of disulfide bond in heavy chain, so that the same class of Ig can be divided into different subclasses (subclasses). For example, human IgG can be classified into IgG1 to IgG4, and IgA can be classified into IgA1 and IgA2. Immunoglobulin (Ig) light chains are classified into kappa (kappa) chains and lambda (lambda) chains according to the light chain structure and antigenicity, whereby Ig can be classified into two types (types), namely kappa type and lambda type.

"Porcine antibodies" comprise amino acid residues from non-porcine HVRs and amino acid residues from porcine FR. In certain embodiments, a swine-derived antibody comprises at least one, and typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to HVRs of a non-swine antibody, and all or substantially all FRs correspond to FRs of a swine antibody. The swine-derived antibody optionally can comprise at least a portion of an antibody constant region derived from a swine antibody. An antibody in a "swine-derived form", such as a non-swine antibody, refers to an antibody that has undergone swine-derived.

The term "polynucleotide" or "nucleic acid" or "nucleotide sequence" as used herein refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral-derived RNA, or plasmid DNA (pDNA). Polynucleotides may comprise conventional phosphodiester linkages or non-conventional linkages (e.g., amide linkages, such 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.

An "antibody fragment" or "antigen-binding fragment" comprises a portion of an intact antibody that still retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, fv, diabodies, triabodies, tetrabodies, cross Fab fragments, linear antibodies, single chain antibody molecules (e.g., scFv), multispecific antibodies formed from antibody fragments and single domain antibodies (single domain antibodies).

The term "antigen binding domain" or "antigen binding site" as used herein refers to the portion of an antigen binding molecule that specifically binds an epitope. More specifically, the term "antigen binding domain" refers to a portion of an antibody that comprises a region that specifically binds to and is complementary to a portion or all of an antigen. In the case of large antigen molecules, the antigen binding molecule may bind only a specific portion of the antigen, which portion is referred to as an epitope. The antigen binding domain may be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, the antigen binding domain is capable of binding to its antigen and blocking or partially blocking the function of said antigen.

The term "epitope" as used herein is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a stretch of contiguous amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids) to which an antigen binding portion binds, thereby forming an antigen binding portion-antigen complex. The antigenic determinants may be present, for example, on the surface of tumor cells, on the surface of microorganism-infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum free material and/or in the extracellular matrix (ECM). Unless otherwise indicated, the protein used as an antigen in the present invention may be any native form of protein of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The antigen may also be a human protein, or the antigen may be a "full length", unprocessed protein, as well as any form of protein resulting from intracellular processing, or a naturally occurring protein variant, such as a splice variant or an allelic variant.

Specific "binding force" or "affinity" of an antibody or antigen-binding fragment thereof to an antigen refers to the strength of non-covalent interactions 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 particular antigen may be measured by an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface Plasmon Resonance (SPR) techniques as well as conventional binding assays; in one embodiment, the extent of binding of an antigen binding molecule to an unrelated protein, e.g., as measured by SPR, is less than about 10% of the extent of binding of the antigen binding molecule to an antigen; the binding affinity may generally be expressed in terms of dissociation constants (KD), in certain embodiments, the dissociation constant (Kd) of the antigen-binding molecule is 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or 0.001nM or less (e.g., 10 ^-7 M or less, e.g., 10 ^-7 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M).

The term "isolated" nucleic acid molecule or polynucleotide as used herein refers to a nucleic acid molecule, DNA or RNA that has been isolated from its natural environment. In the present invention, the recombinant polynucleotide encoding the antibody or antigen-binding fragment thereof contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. An isolated polynucleotide includes a polynucleotide molecule that is normally contained in a cell containing the polynucleotide molecule, but that is present at a chromosomal location that is extrachromosomal or different from its native chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, both in positive and negative strand form, and in double stranded form. The isolated polynucleotides or nucleic acids of the present disclosure further include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid may be or include regulatory elements such as promoters, ribosome binding sites or transcription terminators.

The terms "vector" or "expression vector" and "expression construct" as used herein are used interchangeably to introduce a particular gene into a target cell and direct the expression of a DNA molecule operably linked thereto. The vectors include vectors that are self-replicating nucleic acid structures and that incorporate the genome of a host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can perform transcription of a large number of stable mRNAs. Once the expression vector is within the target cell, ribonucleic acid molecules or proteins encoded by the gene are produced by cellular transcription and/or translation mechanisms. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding an antibody or antigen binding fragment thereof of the invention. The term "expression cassette" according to the invention refers to a recombinantly or synthetically produced polynucleotide having a series of nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette may be introduced into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the invention comprises a polynucleotide sequence encoding an antibody or antigen-binding fragment thereof of the invention.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably herein to refer to cells into which exogenous nucleic acid has been introduced, and include the progeny of such cells. Host cells include "transformants" and "transformed cells," including primary transformed cells and progeny derived therefrom. The nucleic acid of the offspring may not be exactly identical to the parent cell and may contain mutations. The host cell is any type of cell that can be used to produce the bispecific antigen binding molecules of the invention. Host cells include cultured cells, for example cultured mammalian cells such as CHO cells, HEK293 cells, BHK cells, NS0 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 transgenic animals, transgenic plants or cells contained within cultured plant or animal tissues. Host cells are herein not involved in propagation material.

The term "pharmaceutical composition" as used herein means a composition comprising one or more antibodies or antigen-binding fragments thereof of the invention or a mixture of the invention with other chemical components, such as a physiologically/pharmaceutically acceptable carrier or excipient. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The term "pharmaceutically acceptable carrier" as used herein refers to a component of the pharmaceutical composition that is non-toxic to the subject in addition to the active component. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers and/or preservatives.

The term "treatment" as used herein refers to administration of an internally or externally used therapeutic agent, e.g., comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure, or a pharmaceutical composition, to a subject having one or more swine infectious diseases or symptoms caused by African Swine Fever Virus (ASFV) for which the therapeutic agent has a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more diseases or symptoms in the subject individual or population to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

The term "preventing" as used herein refers to delaying, inhibiting or preventing the onset of swine infectious disease caused by African Swine Fever Virus (ASFV) in a subject.

"Percent sequence identity" or "percent identity" 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., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since the gaps are not nucleotides or amino acids, the gaps present in the target sequence are not taken into account. Also, since the target sequence nucleotide or amino acid is counted, and the nucleotide or amino acid from the reference sequence is not counted, gaps in the reference sequence are not counted.

The percent sequence identity can be calculated by determining the number of positions in which the same amino acid residue or nucleobase occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software that is readily available for online use and download. Suitable software programs are available from a variety of sources for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is the bl2seq, which is part of the BLAST suite of programs available from the national center for Biotechnology information, BLAST website (BLAST. Ncbi. Lm. Nih. Gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to make a comparison between 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, part of the EMBOSS suite of bioinformatics programs, and are also available from European Bioinformatics Institute (EBI) on www.ebi.ac.uk/Tools/psa.

The term "conservative substitution" as used herein means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., 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).

Examples and figures are provided below to aid in the understanding of the invention. It is to be understood that these examples and drawings are for illustrative purposes only and are not to be construed as limiting the invention in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention. The reagents and/or kits used in the following examples are all commercially available or can be synthesized by known methods.

The specific conditions not specified in the examples were carried out according to the conventional conditions, the experimental conditions suggested by the manufacturer or the publicly reported conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. The reagent used notes manufacturers, and similar products of other manufacturers have alternatives.

Sequence information

The sequence information involved in the examples of the present invention is shown in table 1:

TABLE 1 sequence information

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.

Example one expression of EP153R protein

1. Construction of recombinant plasmid expressing EP153R

With reference to the EP153R gene sequence of ASFV Chinese epidemic strain Pic/HLJ/2018 (GenBank: MK 333180.1) in GenBank, the nucleotide was codon-optimized and then gene synthesis was performed. The primers are designed respectively, the extracellular segment of EP153R (the amino acid sequence is shown as SEQ ID NO: 63, the nucleotide sequence is shown as SEQ ID NO: 64) is amplified by PCR, a signal peptide (SIGNAL PEPTIDE, SP) is added at the N-terminal (the amino acid sequence is shown as SEQ ID NO: 61, the nucleotide sequence is shown as SEQ ID NO: 62) and a his tag sequence is added at the C-terminal (the amino acid sequence is shown as SEQ ID NO: 65, the nucleotide sequence is shown as SEQ ID NO: 66) and cloned on a eukaryotic expression vector pCMV to construct an expression vector in the form of SP-EP153R Ex-his, and the protein expressed after the transfected cells is used for cutting the signal peptide to form mature recombinant protein which is secreted into a cell culture medium.

Expression of EP153R recombinant proteins

1) Preparation of transfection complexes

A method for preparing a solution of a PEI transfection reagent comprises adding 100mg of a linear PEI transfection reagent (Mw 40000; LABLEAD Co., product catalog number P4000) to 90mL of Milli-Q ultrapure water, stirring uniformly to dissolve completely, adjusting pH to 6.9-7.1, fixing the volume to 100mL with Milli-Q ultrapure water, filtering with a 0.22 μm filter membrane, and collecting the filtrate.

The preparation method of the transfection complex (the dosage of each 1L cell suspension) comprises the steps of adding 1mg of recombinant plasmid (pCMV-SP-EP 153R Ex-his) into 5mL of Hi-exp culture medium (Ao Pu Mai Co., product catalog number: AC 601501) respectively, blowing and mixing uniformly to obtain a liquid phase A, adding 3mL of PEI transfection reagent solution into 5mL of Hi-exp culture medium, blowing and mixing uniformly to obtain a liquid phase B, adding the liquid phase B into the liquid phase A, blowing and mixing uniformly, and standing at room temperature for 5min.

2) Preparation of cell suspensions

293F cells in culture were harvested, after cell counting, centrifuged at 800rpm for 5 minutes, and the supernatant was discarded and resuspended in Hi-exp medium to a cell concentration of 1X 10 ⁶ cells/mL.

3) The transfection complex was added dropwise to 1L of the cell suspension, cultured with shaking at 130rpm for 4-5 days (ambient conditions: 37 ℃ C., 8% CO ₂), then centrifuged at 4000rpm for 20min, the supernatant was collected, and then filtered with a filter membrane having a pore size of 0.45. Mu.m, and the filtrate was collected.

3. Purification of recombinant proteins

Column chromatography (column volume is 10mL; packing is Ni Sepharose 6FF, cytiva (GE life), product catalog number is 17531801), balancing the column with balancing buffer (1 XPBS buffer; 300mM NaCl; pH 7.4), loading the supernatant obtained by the expression in step 2 into the column (loading volume is 1L), loading the column with eluting buffer (1 XPBS buffer; 300mM NaCl;50mM imidazole; pH 7.4) for 5-10 column volumes, and eluting with eluting buffer (1 XPBS buffer; 300mM NaCl;500mM imidazole; pH 7.4) to obtain EP153R recombinant protein.

4. Identification of recombinant proteins

Recombinant proteins were assayed for concentration using nanodrop and identified by SDS polyacrylamide electrophoresis (SDS-PAGE). 1-2 mug of recombinant protein samples were loaded onto a 12% SDS-PAGE gel under denaturing conditions, stained with Coomassie brilliant blue after electrophoresis, and decolorized to reveal clear protein bands, as shown in FIG. 1, since EP153R recombinant proteins were glycosylated, a wider band was exhibited, the band size was between 20-40kDa, and the purity of the purified protein was higher.

Binding of EP153R recombinant proteins to positive serum (ELISA method)

And (3) taking the EP153R recombinant protein, and diluting the recombinant protein with PBS buffer solution until the protein concentration is1 mug/mL, thus obtaining the coating solution. PBST solution PBS buffer containing 0.05% (volume percent) Tween-20. Blocking solution PBST solution containing 0.2g/100mL BSA. The preparation method of the antibody diluent comprises the steps of taking African swine fever positive serum (Chinese veterinary drug administration) as positive serum, taking non-immunized normal pig serum as negative serum control, and diluting the serum 1000 times by using a sealing solution.

The detection is carried out according to the following steps:

1) The 96-well ELISA plate was taken, the coating solution (100. Mu.L/well) was added, incubated at 4℃for 16 hours (overnight), the supernatant was discarded, washed 3 times with PBST solution, and the plate was dried.

2) Taking the 96-well plate with the step 1), adding a blocking solution (200 mu L/well), incubating for 1 hour at room temperature, discarding the supernatant, washing 3 times with PBST solution, and beating to dry.

3) Taking the 96-well plate with the step 2), adding antibody diluent (100 mu L/well), incubating for 1 hour at room temperature, discarding the supernatant, washing 3 times with PBST solution, and beating to dry.

4) Taking the 96-well plate with the step 3), adding HRP-labeled goat anti-pig IgG antibody (Sigma, AP 166P) (100 uL/well for 1-fold dilution), incubating for 1 hour at room temperature, discarding the supernatant, washing 5 times with PBST solution, and beating to dry.

5) Taking the 96-well plate with the step 4), adding TMB color development liquid (100 mu L/well), and reacting for 5-10 minutes in a dark place.

6) The 96-well plate obtained in step 5) was taken, 2M sulfuric acid solution (50. Mu.L/well) was added, and then the absorbance at 450nm (OD ₄₅₀) was measured.

The EP153R recombinant protein can be combined with African swine fever positive serum, and the result is shown in FIG. 2, and the natural conformation of the EP153R protein in the recombinant protein and ASFV can be presumed to have similarity.

Example two hybridoma monoclonal antibody screening

1. Immunized mice:

SPF-class 6-8 week old Balb/c mice (Jiangsu Jiuyaokang biotechnology Co., ltd.) were immunized as follows with the immunogen of EP153R recombinant protein prepared in example one:

Day 1, immunization 1, multiple subcutaneous injections of an immunogen (consisting of 10 μg immunogen, freund's complete adjuvant (Sigma, F5881);

day 29, immunization 2, multipoint subcutaneous injection of an immunogen (consisting of 10 μg immunogen, freund's incomplete adjuvant (Sigma, F5506);

day 57, 3 immunizations, multiple subcutaneous injections of an immunomer (consisting of 10 μg immunogen, freund's incomplete adjuvant);

on day 64, serum was collected from orbital venous blood and used as a test antibody to detect titers by ELISA, and mice with high titers were selected for immunization at the 4 th time and were intraperitoneally injected with 10. Mu.g of immunogen.

2. Hybridoma cell fusion and screening

1) Preparation of feeder cells

The day before fusion, the well developed Balb/c mice were sacrificed at cervical amputation and sterilized in 75% ethanol aqueous solution. 8-10mL of precooled 0.34M sucrose aqueous solution is sucked by a precooled syringe, the needle is injected from the lower right corner peritoneum of the mouse, the needle head does not go out of the peritoneum, and the abdominal cavity is massaged by fingers for about 1 min. Then, the liquid in the abdominal cavity of the mouse was aspirated, added to a pre-chilled 50mL centrifuge tube, pre-chilled 1640 complete medium was added, centrifuged at 1500rpm for 5min, the supernatant was discarded, the cell pellet was resuspended in 1640 complete medium (HAT medium) containing HAT (Sigma, cat# H0262) to a cell concentration of 1X 10 ⁵ cells/mL, and then the cell suspension was added to a 96-well cell culture plate (100. Mu.L/well) for culturing.

2) Preparation of spleen cells

After 4 days of the completion of the 4 th immunization in step 1, mice were sacrificed by eye-catching, and blood and spleen were collected, respectively. Serum was isolated from blood and used as an EP153R immunopositive serum control. A10 cm diameter petri dish was taken, 10mL 1640 medium and DNase (final concentration 5. Mu.g/mL) were added, then the mouse spleen was added, ground and blown down as a single cell suspension, filtered using a 70 μm filter screen and the filtrate was collected into a 50mL centrifuge tube, centrifuged at 1500rpm for 5min, and the supernatant was discarded. Cell pellet was resuspended in ACK erythrocyte lysate, incubated at room temperature for 2min, cells were washed with 1640 medium, the supernatant was centrifuged off, cells were resuspended in 20mL 1640 medium, and counted after pipetting.

3) Cell fusion

SP2/0 mouse myeloma cells in good condition and in logarithmic growth phase were collected and washed with 1640 medium. SP2/0 mouse myeloma cells and spleen cells were mixed in a ratio of 1:1-1:3, centrifuged at 1500rpm for 10min, and the supernatant was discarded. The cells were beaten to paste, then placed in a 37 ℃ water bath, 1mL of a pre-heated 50% PEG solution was added dropwise, 40mL of pre-heated 1640 medium was slowly added, centrifuged at 1500rpm for 10min, and the supernatant was discarded. The cell pellet was taken apart, then 10mL of HAT medium was added and blown up and down several times, and then HAT medium was added to about 90mL, mixed well and added dropwise to a feeder cell culture plate, 2 drops per well, and placed in a 37℃cell incubator for cultivation.

4) Screening of hybridoma cell positive wells

Half-volume replacement of HAT medium was performed 4 days after cell fusion, and hybridoma cells grew to a certain size for about 7-10 days, about 200. Mu.L of medium was aspirated the day before detection, and 200. Mu.L of fresh 1640 complete medium (HT medium) containing HT (Sigma, cat. H0137) was added again. On the day of detection, a culture solution of hybridoma cell clusters is taken as a test antibody, EP153R immune positive serum is taken as a positive control, non-immune mouse serum is taken as a negative control, and positive clones are screened by detecting the binding of the EP153R recombinant protein through an ELISA method.

5) Subcloning of positive hybridoma cells

The method comprises the steps of performing first subcloning on a HT culture medium, selecting positive clone holes for first subcloning, observing cell states and cell mass sizes under a microscope, gently blowing and mixing cells needing subcloning under aseptic conditions, avoiding blowing out bubbles, sucking 10 mu L of counting cells, adding 10 mu L of 0.04% trypan blue solution into the counted cells, counting 8 big lattice cell numbers after mixing uniformly, calculating cell concentration, sucking 100-150 cells into 9.5mL of culture medium through a limiting dilution method according to the cell counting result, mixing uniformly, and then dripping into a feeder cell culture plate for 1 day of culture.

Subcloning for about 5 days, counting hybridoma clones in each hole under an inverted microscope, detecting again when hybridoma cells grow to a proper size, screening positive monoclonal cells for secondary subcloning, changing a culture medium into a 1640 complete culture medium, and continuing subcloning for 2-3 times until the obtained monoclonal hybridoma cell strain can stably secrete required antibodies.

6) Expanded culture and cryopreservation of hybridoma cells

① The positive hybridoma obtained by identification is transferred to a 24-hole cell culture plate for amplification culture until the confluency reaches about 80%.

② After step ① was completed, the cells were transferred to a T25 cell culture flask and cultured until the cell confluence reached about 80%.

③ After the step ② is completed, repeatedly blowing the inner wall of the culture flask with a culture medium, transferring the culture flask into a sterile centrifuge tube, centrifuging at 1500rpm for 5min, discarding the supernatant, re-suspending the cells with 3mL of cell freezing solution, transferring the cell suspension into the freezing tube after fully and uniformly mixing, transferring the cell suspension into a program cooling box, standing at-80 ℃ for 24h, and transferring the cell suspension into liquid nitrogen for long-term storage.

Based on the above procedure, a plurality of hybridoma cells secreting the target mab (i.e., monoclonal antibody that specifically binds to EP153R protein) including monoclonal HY002 (derived from 9D9 clone) contained in the present invention were obtained (fig. 3).

EXAMPLE three preparation and identification of monoclonal antibodies

1. Preparation of ascites

1) On day 0, 500. Mu.L of liquid paraffin was intraperitoneally injected into NCG mice (Jiangsu Jiuyaokang biotechnology Co., ltd.), which were preferably mice produced by a donor or larger individuals.

2) The HY002 hybridoma cells in the logarithmic growth phase were collected at 9-11 days, collected in a centrifuge tube, centrifuged at 1500rpm for 5 minutes, the supernatant was discarded, the cells were gently sprung, 40mL of PBS was added to wash the cells, centrifuged at 1500rpm for 5 minutes, the supernatant was discarded, the washing was repeated once and counted, PBS was added to prepare a cell suspension of 1X 10 ⁶-2×10⁶ cells/mL, and 500. Mu.L of each mouse was intraperitoneally injected.

3) Taking out ascites according to the state of the mice on about day 19-23, centrifuging at 12000rpm for 10 minutes, sucking out pale yellow ascites, and preserving at-80 ℃ for standby.

2. Purification of antibodies

Antibodies were purified from ascites fluid using an affinity column (packing protein At Beads LX, product catalog No. SA08501L, all-day-and-earth-and-biotechnology limited).

1) The column was equilibrated with PBS buffer.

2) Taking out the ascites 12000rpm, centrifuging for 10 minutes, taking out the supernatant, diluting the supernatant by 5 times with PBS buffer solution, filtering the supernatant by 0.45 mu m, and loading the filtered ascites diluent on a chromatographic column.

3) After all ascites had been completed, 10-15 column volumes were washed with PBS buffer.

4) The column-passing solution was eluted with glycine buffer (pH 2.5-3.0,0.1M) and mixed with 1 part by volume of neutralization buffer (i.e., tris-HCl buffer, pH9.0, 1M) to give a mixed solution.

5) Concentrated solution exchange was performed using a 30K ultrafiltration tube (Millipore, UFC 903096), and the buffer system was replaced with PBS buffer to obtain purified monoclonal antibodies, and the SDS-PAGE patterns were shown in FIG. 4.

3. Identification of monoclonal antibody subtypes

And diluting the HY002 monoclonal antibody into 1 mug/mL of working solution. The antibody subtype was identified using the monoclonal antibody subtype identification kit and the procedure was performed according to instructions. The monoclonal antibody subtype identification kit (Isotyping Kit for Mouse Monoclonal Antibody) is SEK003, product catalog number of Beijing Yiqiao Shenzhou technology and technology Co., ltd.

The results are shown in FIG. 5, HY002 is the IgG2a subtype.

Example IV specific binding of monoclonal antibodies to EP153R protein

1. To achieve detection of binding of mab to protein by flow cytometry, recombinant plasmids in the form of "pTT3-Flag-EP153R-HA-IRES-GFP" were constructed on the engineered pTT3-IRES-GFP vector by adding a Flag tag sequence to the N-terminus of EP153R and an HA tag sequence to the C-terminus, and the amino acid and nucleotide sequences of Flag-EP153R-HA and GFP are shown in Table 1.

2. The recombinant plasmids described above were transfected into 293T cells (6 cm dish) with PEI transfection reagent, respectively. 24h after transfection, the cells were digested into single cell suspensions with 2mM EDTA. 2mL of FACS buffer was added, the cells were washed by centrifugation 5min at 1500 rpm and the supernatant was discarded. Cells were divided into two portions, one portion was added to FACS buffer (1 XPBS+0.2% BSA+2mM EDTA) and the other portion was lysed with RIPA and then boiled at 100℃for 10 minutes with 5X SDS loading buffer (containing 5% beta-mercaptoethanol).

3. Cells added with FACS buffer were divided into three portions, centrifuged at 1500 rpm for 5 min, and the supernatant discarded. 100. Mu.L of antibody working solution diluted to 1. Mu.g/mL with FACS buffer was added, and incubated on ice for 30 minutes as anti-flag (Sigma, F1804), anti-HA (Abcam, ab 18181) and HY002, respectively. Cells were washed once with FACS buffer and the supernatant was discarded by centrifugation. mu.L of 700-fold diluted PE-goat anti-mouse IgG (BioLegend, 405307) was added separately and stained in ice protected from light for 30: 30 min. Cells were washed once with FACS buffer and the supernatant was discarded by centrifugation. 400 μl of 2% PFA resuspended cells were added to each sample and the samples were analyzed by flow cytometry and the results were analyzed by FlowJo software.

As a result, as shown in FIG. 6, since the surface staining of the membrane was performed, the staining result of the Flag antibody was negative, the staining result of the HA antibody was positive, and it was confirmed that EP153R was a characteristic of a class II transmembrane protein, the C-terminal thereof was located outside the cell membrane, and HY002 antibody was able to specifically bind to the extracellular portion of EP 153R.

4. Samples prepared in 2 were subjected to SDS-PAGE electrophoresis after thermal denaturation, and proteins were transferred to PVDF membrane, and then blocked with blocking solution (5 g of skimmed milk powder was dissolved in 100mL of TBST buffer, TBST buffer: 1 XTBS+0.1% Tween 20) at room temperature for 1 hour. A solution of HY002 antibody diluted to 1. Mu.g/mL with blocking solution was added and incubated on a shaker at room temperature for 1-2 hours or overnight at 4 ℃. The mixture was thoroughly washed with TBST on a shaker at room temperature for 10min each for 3 times. HRP-labeled goat anti-mouse IgG (Zhonghuperzia ZB-2305) was diluted 1:10000-fold with blocking solution and incubated in a shaker for 1 hour at room temperature. The mixture was thoroughly washed with TBST on a shaker at room temperature for 10min each for 3 times. The PVDF membrane was developed.

As shown in FIG. 7, HY002 can be used for Western Blot detection and can specifically bind to EP153R protein.

Example five binding of HY002 monoclonal antibody to ASFV virus-infected cells

1. Porcine alveolar macrophages (PAM cells, manufactured by Jin Yubao m biopharmaceutical limited, provided) infected with ASFV (HLJ/18) in 96-well plates for 72h were used as negative control for immunohistochemical staining, with specific staining steps as follows:

1) Removing 96-well plate liquid, adding 200 μl/well of 10% neutral formalin fixation solution, fixing at room temperature for 30min, removing 96-well plate liquid, adding 200 μl/well permeabilization solution (500mL DPBS+5mL TritonX-100), washing for 2 times, and soaking for 5min each time.

2) Discarding 96-well plate liquid, filling cell holes with antigen retrieval liquid (10.5 g of citric acid and 14.19g of disodium hydrogen phosphate are dissolved to 500mL with purified water), putting the mixture into a water bath kettle at the water temperature of 60 ℃, taking out the mixture after the water is opened for 2min, naturally cooling the mixture for 10-20 min, discarding 96-well plate liquid, adding 200 mu L/well permeabilization liquid, washing the mixture for 2 times, and soaking the mixture for 5min each time.

3) Discarding 96-well plate liquid, diluting HY002 monoclonal antibody to 8 μg/mL 100 μl/well with PBS, adding 96-well plate, incubating at 37deg.C for 30min, discarding 96-well plate liquid, adding 200 μl/well permeabilization liquid, washing for 2 times, and soaking for 5min each time.

4) The 96-well plate liquid was discarded, goat anti-mouse IgG secondary antibody (Zhonghua gold bridge ZB-2305) was labeled with HRP, incubated at 37℃for 30min, the 96-well plate liquid was discarded, 200. Mu.L/well permeabilization solution was added, and washing was performed 2 times, each time for 5min.

5) The 96-well plate liquid is discarded, 100 mu L/well of DAB developing solution (Abies sinensis golden bridge biotechnology Co., ltd.) with working concentration is added, the color development is carried out for 10min at room temperature, the developing solution is discarded, 200 mu L/well PBS is washed for 2 times, and finally the solution is not discarded.

6) The cells were observed and recorded as shown in FIG. 8A, the left panel shows non-inoculated blank cells without specific staining, and the right panel shows ASFV virus-inoculated cells with specific staining.

2. After lysis of ASFV-infected cells and negative control PAM cells as described above with RIPA, they were cooked at 5X SDS loading buffer 100 ℃for 10 min and denatured at high temperature. The result of western blot verification shows that HY002 can be specifically bound to the corresponding band in ASFV infected cells (FIG. 8B).

Example six obtaining of HY002 monoclonal antibody variable region sequence

1X 10 ⁶-1×10⁷ HY002 hybridoma cells were centrifuged at 1500rpm for 5min, the supernatant was discarded, washed with PBS, centrifuged again to remove the supernatant, 1mL of pre-chilled Trizol (Invitrogen, 15596018 CN) was added, and RNA was extracted according to the protocol. Reverse transcription into cDNA was performed using oligo dT primer and M-MLV reverse transcriptase (Progema, M1705).

Using cDNA as a template, primers directed against the antibody variable region, the mouse VH sequence upstream primers (shown as SEQ ID NOS: 7-18 in Table 1) were paired with the subtype-opposite heavy chain downstream primers (shown as SEQ ID NOS: 19 in Table 1), the mouse V kappa sequence upstream primers (shown as SEQ ID NOS: 20-30 in Table 1) were paired with the light chain downstream primers (shown as SEQ ID NOS: 31 in Table 1), and PCR amplification was performed using PRIMESTAR MAX DNA polymerase (TaKaRa, R045), the amplification system and the PCR reaction procedure were as shown in tables 2 and 3 below.

TABLE 2 PCR amplification System

TABLE 3 PCR reaction procedure

And (3) performing 1% agarose gel electrophoresis analysis on the PCR amplified product, cutting out specific strips with correct size in the amplified product, recovering the gel product, sending to a sequencing company for sequencing to obtain the gene sequence of the heavy chain variable region of the antibody and the gene sequence of the light chain variable region of the antibody, and further obtaining the amino acid sequence of the heavy chain variable region of the antibody and the amino acid sequence of the light chain variable region of the antibody.

The amino acid sequence of the HY002 antibody heavy chain variable region is shown as SEQ ID NO. 32, the nucleotide sequence of the antibody heavy chain variable region is shown as SEQ ID NO. 34, the amino acid sequence of the antibody light chain variable region is shown as SEQ ID NO. 33, and the nucleotide sequence of the antibody light chain variable region is shown as SEQ ID NO. 35. The CDR amino acid sequences of the HY002 antibodies are shown in tables 4 and 5.

TABLE 4 CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of HY002 antibodies

TABLE 5 CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of HY002 antibodies

Example seven preparation of genetically engineered antibodies

1. Preparation of recombinant expression vector for genetically engineered antibody

Cloning and recombining a signal peptide sequence (the amino acid sequence is shown as SEQ ID NO: 61, the nucleotide sequence is shown as SEQ ID NO: 62), an HY002 heavy chain variable region sequence and a mouse heavy chain constant region sequence (the amino acid sequence of a mouse heavy chain (IgG 2a subtype) constant region is shown as SEQ ID NO: 57, and the nucleotide sequence is shown as SEQ ID NO: 59) onto a PTT3 vector to obtain enHY002 heavy chain recombinant expression plasmid. Cloning and recombining a signal peptide sequence (the amino acid sequence is shown as SEQ ID NO: 61, the nucleotide sequence is shown as SEQ ID NO: 62), an HY002 light chain variable region sequence and a mouse light chain constant region sequence (the amino acid sequence of a mouse light chain (Ig kappa subtype) constant region is shown as SEQ ID NO: 58, and the nucleotide sequence is shown as SEQ ID NO: 60) onto a PTT3 vector to obtain the enHY002 antibody light chain recombinant expression plasmid. 293F cells were co-transfected with recombinant heavy and light chains 1:1 using PEI transfection reagent as described in example one and incubated for 6 days to obtain antibodies secreted in the medium. The antibodies in the medium were purified as described in example three to give the genetically engineered antibody enHY002 in high purity. The specificity and antibody titer of enHY were determined by ELISA method, and the results are shown in FIG. 9, wherein the A graph shows that the purity of the genetically engineered antibody enHY002 is consistent with the light and heavy chain band size and HY002, the B graph shows that the binding reaction between the two proteins of HY002 and enHY and EP153R begins at about 10ng/mL when the two proteins are respectively combined by gradient dilution, and the titers of the two proteins are not obviously different.

The above results show that the variable region sequences measured by HY002 can be used for the preparation of genetically engineered antibodies that bind to the EP153R protein.

The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.

Claims (14)

1. An antibody or antigen binding fragment thereof that specifically binds to the EP153R protein of African swine fever virus, wherein,

The antibody or antigen binding fragment thereof comprises the complementarity determining regions:

(a1) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region shown in SEQ ID NO. 32 and/or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region shown in SEQ ID NO. 33;

(a2) CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region and/or CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region described below, wherein at least one CDR contains a mutation, preferably a substitution, deletion or addition of one or several amino acids, preferably a conservative substitution, compared to the heavy chain variable region and/or light chain variable region described in (a 1).

2. The antibody or antigen-binding fragment thereof according to claim 1,

The complementarity determining regions are defined according to Kabat, chothia, IMGT, contact, or AbM numbering system;

Preferably, the antibody or antigen binding fragment thereof comprises the heavy chain variable region and/or light chain variable region, wherein the complementarity determining regions are defined according to the IMGT numbering system:

A heavy chain variable region comprising 3 CDRs of CDR-H1 of SEQ ID NO. 36 or a variant thereof, CDR-H2 of SEQ ID NO. 37 or a variant thereof, CDR-H3 of SEQ ID NO. 38 or a variant thereof, and/or a light chain variable region comprising 3 CDRs of CDR-L1 of SEQ ID NO. 48 or a variant thereof, CDR-L2 of SEQ ID NO. 49 or a variant thereof, CDR-L3 of SEQ ID NO. 50 or a variant thereof;

preferably, the antibody or antigen binding fragment thereof comprises the heavy chain variable region and/or the light chain variable region, wherein the complementarity determining regions are defined according to the Kabat numbering system:

A heavy chain variable region comprising 3 CDRs of CDR-H1 of SEQ ID NO 39 or a variant thereof, CDR-H2 of SEQ ID NO 40 or a variant thereof, CDR-H3 of SEQ ID NO 41 or a variant thereof, and/or a light chain variable region comprising 3 CDRs of CDR-L1 of SEQ ID NO 51 or a variant thereof, CDR-L2 of SEQ ID NO 52 or a variant thereof, CDR-L3 of SEQ ID NO 53 or a variant thereof;

Preferably, the antibody or antigen binding fragment thereof comprises the following heavy chain variable region and/or light chain variable region, wherein the complementarity determining regions are defined according to the Chothia numbering system:

a heavy chain variable region comprising 3 CDRs of CDR-H1 of SEQ ID NO. 42 or a variant thereof, CDR-H2 of SEQ ID NO. 43 or a variant thereof, CDR-H3 of SEQ ID NO. 44 or a variant thereof, and/or a light chain variable region comprising 3 CDRs of CDR-L1 of SEQ ID NO. 51 or a variant thereof, CDR-L2 of SEQ ID NO. 52 or a variant thereof, CDR-L3 of SEQ ID NO. 53 or a variant thereof;

Preferably, the antibody or antigen binding fragment thereof comprises the heavy chain variable region and/or light chain variable region, wherein the complementarity determining regions are defined according to the Contact numbering system:

A heavy chain variable region comprising 3 CDRs of CDR-H1 of SEQ ID NO 45 or a variant thereof, CDR-H2 of SEQ ID NO 46 or a variant thereof, CDR-H3 of SEQ ID NO 47 or a variant thereof, and/or a light chain variable region comprising 3 CDRs of CDR-L1 of SEQ ID NO 54 or a variant thereof, CDR-L2 of SEQ ID NO 55 or a variant thereof, CDR-L3 of SEQ ID NO 56 or a variant thereof;

wherein the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, preferably the substitution is a conservative substitution.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises:

A heavy chain variable region comprising a sequence as set forth in SEQ ID NO. 32 or a variant thereof and/or a light chain variable region comprising a sequence as set forth in SEQ ID NO. 33 or a variant thereof;

Wherein 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 to the sequence from which it is derived, or has a substitution, deletion, or addition of one or more amino acids to the sequence from which it is derived, preferably the substitution is a conservative substitution.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, or a swine-derived antibody;

Preferably, the antibody is of the IgA, igD, igE, igG or IgM type, more preferably the antibody is of the IgG1, igG2, igG3, igG4, igA1 and IgA2 types.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, further comprising a constant region derived or derived from a murine immunoglobulin, or a porcine immunoglobulin;

Preferably, the heavy chain of the antibody or antigen binding fragment thereof comprises a heavy chain constant region derived or derived from a murine immunoglobulin;

Preferably, the antibody or antigen binding fragment thereof comprises a wild-type Fc region, or comprises a mutated or chemically modified Fc region having altered effector function compared to the wild-type Fc region;

Preferably, the antibody or antigen binding fragment thereof comprises a heavy chain constant region derived or derived from murine IgG2 a;

preferably, the light chain of the antibody or antigen binding fragment thereof comprises a light chain constant region derived or derived from a murine immunoglobulin;

Preferably, the antibody or antigen binding fragment thereof comprises a light chain constant region derived or derived from murine igkappa;

preferably, the antibody or antigen binding fragment thereof comprises a heavy chain constant region as shown in SEQ ID NO. 57 or a variant thereof having a conservative substitution of up to 20 amino acids compared to SEQ ID NO. 57;

preferably, the antibody or antigen binding fragment thereof comprises a light chain constant region as shown in SEQ ID NO. 58 or a variant thereof having a conservative substitution of up to 20 amino acids compared to SEQ ID NO. 58;

Preferably, the antibody or antigen binding fragment thereof comprises a heavy chain constant region as set forth in SEQ ID NO. 57 and a light chain constant region as set forth in SEQ ID NO. 58.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises:

A heavy chain comprising a heavy chain variable region as set forth in SEQ ID NO. 32 and a heavy chain constant region as set forth in SEQ ID NO. 57, and a light chain comprising a light chain variable region as set forth in SEQ ID NO. 33 and a light chain constant region as set forth in SEQ ID NO. 58.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is ScFv, fab, fab ', fab ' -SH, F (ab ') 2, fv fragment, disulfide-linked Fv, diabody, bispecific antibody, or multispecific antibody;

Preferably, the antibody or antigen binding fragment thereof is labeled;

preferably, the antibody or antigen binding fragment thereof is provided with a detectable label.

8. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof, heavy and/or light chain thereof, heavy chain variable region thereof, and/or light chain variable region thereof of any one of claims 1 to 7;

Preferably, the isolated nucleic acid molecule comprises a nucleic acid molecule encoding a heavy chain variable region of an antibody comprising (i) a nucleotide sequence as set forth in SEQ ID NO: 34, (ii) a sequence substantially identical to SEQ ID NO: 34, or (iii) a degenerate sequence as set forth in (i) or (ii) above, and/or a nucleic acid molecule encoding a light chain variable region of an antibody comprising (iv) a nucleotide sequence as set forth in SEQ ID NO: 35, (v) a sequence substantially identical to SEQ ID NO: 35, or (vi) a degenerate sequence as set forth in (iv) or (v) above.

9. A vector comprising the nucleic acid molecule of claim 8, preferably said 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 of making the antibody or antigen-binding fragment thereof of any one of claims 1 to 7, comprising culturing the host cell of claim 10 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.

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

13. A test kit comprising an antibody or antigen binding fragment thereof according to any one of claims 1 to 7, or a nucleic acid molecule according to claim 8, or a vector according to claim 9, or a host cell according to claim 10, for use in detecting the presence or level of african swine fever virus or african swine fever virus EP153R protein in a sample.

14. Use of the antibody or antigen binding fragment thereof of any one of claims 1 to 7, or the nucleic acid molecule of claim 8, or the vector of claim 9, or the host cell of claim 10, or the pharmaceutical composition of claim 12, in the preparation of a reagent for one or more of the following:

1) Detecting the presence or level of african swine fever virus or african swine fever virus EP153R protein in the sample;

2) Preventing and/or treating diseases related to African swine fever virus;

Preferably, the African swine fever virus-related disease is African swine fever.