CN115838416A

CN115838416A - Antibody for African swine fever CD2v and application thereof

Info

Publication number: CN115838416A
Application number: CN202211644186.5A
Authority: CN
Inventors: 黄亚杰; 魏化伟
Original assignee: Jiangsu Dongkang Biomedical Technology Co ltd
Current assignee: Jiangsu Dongkang Biomedical Technology Co ltd
Priority date: 2022-12-20
Filing date: 2022-12-20
Publication date: 2023-03-24

Abstract

The invention belongs to the field of biological medicines, and particularly relates to an antibody against African swine fever CD2v and application thereof. Specifically, the invention provides an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises light chain CDR1-3 and heavy chain CDR1-3, wherein the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO.2, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.3, the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.4, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.5, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.6, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO. 7.

Description

Antibody for African swine fever CD2v and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an antibody against African swine fever CD2v and application thereof.

Background

African Swine Fever (ASF) is an acute, hemorrhagic and virulent infectious disease caused by African Swine Fever Virus (ASFV) infecting domestic pigs and various wild pigs (such as African wild pigs and European wild pigs). The world animal health Organization (OIE) classifies the animal epidemic disease as a legal report animal epidemic disease, and the disease is also a type of animal epidemic disease which is mainly prevented in China. The clinical symptoms of African swine fever are similar to swine fever symptoms, and the clinical diagnosis can only be determined by means of laboratory monitoring.

ASFV is a nucleoplasmic double-stranded DNA large virus, which is an icosahedral structure and coated by an envelope, the genome sizes of different strains are slightly different, the length is between 170 and 190kb, more than 150 main open reading frames are provided, about 200 proteins are coded, and a plurality of proteins related to virus replication, immune escape, virus transmission and the like exist, so that the virus is mainly detected by an ELISA (enzyme-linked immuno sorbent assay) method and a fluorescence PCR (polymerase chain reaction) method at present.

About 50 structural proteins coded by ASFV genes comprise cyst membrane proteins CD2v, p30 and p54, capsid proteins p72 and p49, nucleocapsid protein polyprotein precursors pp20 and pp62, and DNA binding proteins p10 and pA104R. The envelope protein CD2v is encoded by the EP402R gene, also known as pEP402R, and is approximately 45.3kD in protein size, and is designated CD2v because the amino acid sequence encoding the extracellular domain immunoglobulin-like domain of the protein is very similar to CD2 of the host cell. The protein is a glycoprotein, can help ASFV infected cells to combine with erythrocytes in pig blood, and promote the spread of virus in a host. CD2v plays an important role in promoting virus replication and transmission, virus immune escape and the like, and is a hot target for ASFV vaccine research. Through bioinformatics analysis of CD2v, specific antigen epitopes exist in the sequence, and the sequence has potential to be used as an antigen.

Disclosure of Invention

The present disclosure provides an antibody or antigen-binding fragment thereof, which is capable of specifically binding to a CD2v protein, with high affinity for CD2v. In addition, the disclosure also provides methods of making and/or using the antibodies or antigen-binding fragments thereof.

Product(s)

In a first aspect, the invention provides an antibody or antigen-binding fragment thereof comprising light chain CDRs 1-3 and heavy chain CDRs 1-3,

wherein, the amino acid sequence of the heavy chain CDR1 is shown as SEQ ID NO.2, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.3, the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO.4,

the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.5, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.6, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 7.

In some embodiments, the antibody comprises a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a bispecific antibody.

In some embodiments, the antigen binding fragment comprises a Fab fragment, a Fab' fragment, a F (ab) ₂ Fragments, fv fragments and ScFv.

In some embodiments, the CD2v comprises wild-type CD2v or a mutant thereof.

In some embodiments, the heavy chain variable region of the antibody is as set forth in SED ID No. 8.

In some embodiments, the variable region of the light chain of the antibody is as set forth in SED ID No. 9.

In some embodiments, the antibody further comprises a linker sequence, which may be (GGGGS) n, (GGGS) n, (SSSSG) n, (gsgsgsa) n, and (GGSGG) n. Specifically, "GGGGS" refers to the amino acid sequence Gly-Gly-Gly-Gly-Ser, "GGGS" refers to the amino acid sequence Gly-Gly-Gly-Ser, "SSSSSG" refers to the amino acid sequence Ser-Ser-Ser-Gly, "GSGSA" refers to the amino acid sequence Gly-Ser-Gly-Ser-Ala, and "GGSGG" refers to the amino acid sequence Gly-Gly-Ser-Gly-Gly. And n is an integer of 1 or more.

Preferably, the antibody further comprises a constant region.

Preferably, the constant region comprises a constant region of any origin, more preferably, the constant region is a human antibody constant region.

Preferably, the constant regions of the human antibody include heavy chain constant regions of human IgG1, igG2, igG3 or IgG4, light chain constant regions of kappa or lambda type, and the like.

The "CD2v", "CD2v protein" according to the invention includes african swine fever CD2v protein and mutants thereof, which have one or more mutations relative to the CD2v protein wild type. The envelope protein CD2v is encoded by the EP402R gene, the total length of the gene is 1083bp, and the size of the encoded protein is 45.3kD, so that the CD2v is also called pEP402R.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody provided herein and the CD2v protein to which it is directed.

In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof.

Preferably, the nucleic acid molecule comprises DNA, RNA or hybrids of DNA and RNA, which may be double-stranded or single-stranded.

In another aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid molecule encoding an antibody or antigen-binding fragment thereof.

As used herein, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed. The genetic material component carried in the vector may be expressed in the host cell by transforming, transducing or transfecting the host cell with the vector.

Preferably, the vector is an expression vector.

Preferably, the vector comprises a bacterial plasmid vector, a bacteriophage vector, a yeast plasmid vector, an adenoviral vector, a retroviral vector or a lentiviral vector.

Preferably, the vector may be a commercially available vector or a vector constructed by itself as needed, and the method for constructing the vector is well known in the art.

In some embodiments, the vector may comprise a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain an origin of replication, but does not include only these.

In another aspect, the present disclosure provides a cell comprising one or more of the foregoing antibodies or antigen-binding fragments thereof, nucleic acid molecules, or vectors.

Preferably, the cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

As used herein, the cell can be used to proliferate and/or express a nucleic acid. Such cells include, but are not limited to, prokaryotic cells, including, but not limited to, strains of aerobic, microaerophilic, capnophilic, facultative, anaerobic, gram-negative and gram-positive bacterial cells, such as the following strains and derivatives thereof: coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis), bacteroides fragilis (Bacteroides fragilis), clostridium perfringens (clostridium perfringens), clostridium difficile (clostridium difficile), corynebacterium crescentum (Caulobacter creescens), lactococcus lactis (Lactococcus lactis), bacterium demethylum (Methylobacterium extorquens), diplococcus mannii (Neisseria menngiensis), diplococcus meningitidis (Neisseria meningitidis), pseudomonas fluorescens (Pseudomonas fluorescens) and Salmonella typhimurium (Salmonella typhimurium); and eukaryotic cells, including but not limited to yeast strains, such as the following strains and derivatives thereof: pichia pastoris (Pichia pastoris), pichia methanolica (Pichia methanolica), pichia angusta (Pichia angusta), schizosaccharomyces pombe (Schizosaccharomyces pombe), saccharomyces cerevisiae (Saccharomyces cerevisiae) and Yarrowia lipolytica (Yarrowia lipolytica); insect cells and cell lines derived from insects such as Spodoptera frugiperda (Spodoptera frugiperda), trichoplusia ni (Trichoplusia ni), drosophila melanogaster (Drosophila melanogaster), and tobacco hornworm (Manduca sexta), among others; and mammalian cells and cell lines derived from mammalian cells, such as mice, rats, hamsters, pigs, cows, horses, primates, and humans, among others.

Exemplary cells that can be used in the present invention include, but are not limited to: CHO cells, 293 cells, CHO-K1 cells, HEK293 cells, caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, jurkat cells, hepG2 cells, heLa cells, HT-1080 cells, HCT-116 cells, hu-h7 cells, huvec cells, molt 4 cells and the like.

In another aspect, the present invention provides a kit for detecting CD2v protein, wherein the kit comprises any one or more of the antibody or antigen-binding fragment, the nucleic acid molecule, the vector, and the host cell.

Preferably, the kit may comprise other materials, such as cell lysates, stabilizers, buffers, packaging materials, or instructions for use.

Applications of

In another aspect, the present invention provides the use of any one of the aforementioned antibody or antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned host cell, and the aforementioned kit for detecting CD2v protein. Or, the application in the preparation of products for detecting CD2v.

Production method

In another aspect, the present disclosure provides a method of producing the aforementioned antibody or antigen-binding fragment thereof, comprising expressing a nucleic acid encoding the same.

More specifically, cells are cultured in an environment that allows expression of the nucleic acid, which cells contain a nucleic acid encoding the aforementioned antibody or antigen-binding fragment thereof.

Such expression of the encoding nucleic acid can be accomplished by means conventional in the art.

Preferably, the encoding nucleic acid is transferred into the cell by a method dependent on the cell type. For example, for prokaryotic cells, calcium chloride transfection is commonly used, while for other cellular hosts, calcium phosphate treatment, electroporation, lipofection, biolistic or virus-based transfection may be used. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection.

In some embodiments, the method optionally further comprises the step of collecting, purifying the antibody or antigen-binding fragment thereof.

Preferably, the collection and purification may be performed by a method commonly used in the art. For example, the medium and host cells are separated by centrifugation, cells are disrupted by high-pressure homogenization, cell debris is removed by centrifugation, and the antibody is purified by affinity chromatography. For the isolation and purification of the resulting product, purity identification can be performed using a method commonly used in the art. For example, coomassie blue method, kjeldahl method, biuret method, lowry method, ultraviolet absorption method, affinity chromatography, antigen-antibody method, electrophoresis (for example, sodium dodecyl sulfate polyacrylamide gel electrophoresis), sedimentation analysis, diffusion analysis, isotachy method, protein mass spectrometry, and the like.

Detection method

In another aspect, the present invention also provides a method for detecting CD2v, the method comprising contacting a substance to be detected with any one of: the antibody or the antigen binding fragment thereof, the nucleic acid molecule, the vector, the cell and the kit are provided.

In one embodiment, the method is performed in vitro.

In one embodiment, the method is for non-diagnostic purposes.

Preferably, the test substance comprises a sample which may be from an animal, e.g. bovine, ovine, porcine, equine, donkey.

Preferably, the sample may be a nasal swab, purulent nasal fluid, genital swab, semen, respiratory mucosa, part of tonsil, lung, bronchial lymph node, animal fetus, liver, lung, kidney, and placental cotyledon.

Preferably, the analyte comprises an environmental sample that may also be derived from a farm, such as air, water, soil, farm equipment, and the like.

Preferably, the method of detection may be combined with any one of: enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography and other detection methods.

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

FIG. 1 is an electrophoretogram for detecting monoclonal antibody 5D 8.

FIG. 2 is an HPLC chart for detecting monoclonal antibody 5D 8.

FIG. 3 is a graph showing the binding activity of monoclonal antibody 5D8 detected by ELISA.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 screening of monoclonal antibodies

1. Recombinant expression of immunogens

Synthesizing an African swine fever CD2v protein sequence, and constructing to a pEM5.1 vector; extracting plasmids for transfection; transfecting to HEK293 cells, and culturing the cells for 7 days; and (4) harvesting the supernatant, purifying by using a Ni column, and concentrating and replacing a buffer solution to obtain the recombinant African swine fever CD2v protein.

The sequence of the recombinant African swine fever CD2v protein is derived from Uniprot, and the sequence information is as follows: NIIIWSTLNQTV FLNNIFTINDTYGGLFWNTYYDNNRSNFTYCGIAGNYCSCCGHNISLYNTTNNCSLIIFPNNTEIFNRTYELVYLDKKINYTVKLLKSVDSPTITYNCTNSLITCKNNNGTNVNIYLIINNTIVNDTNGDILNYYWNGNNNFTATCMINNTISSLNETENINCTNPILKYQNYLSTGSHHHHHH (shown in SEQ ID NO. 1).

2. Immunization

Mice were immunized, fused SP2/0, and subclones were screened.

The method comprises the following specific steps: the first immunization is carried out by 100 mug each of Freund's complete adjuvant through intraperitoneal injection, the total dose is 0.5 ml/mouse, and the second immunization is carried out at intervals of 3 weeks; taking Freund incomplete adjuvant at a dose of 50 μ g/0.5 ml/piece for the second time, and performing the third immunization at an interval of 2 weeks; cell fusion was prepared 10 days after the third injection; taking feeder cells, and pressing 10 ⁵ Perforamen, plating 10 the day before fusion ⁵ Each cell is 100 mu l/hole; mice immunized spleen cells were fused with prepared myeloma cells using the fusion agent PEG, and plated on 96-cell culture plates to which feeder cells had been added at 100. Mu.l/well.

3. Screening and cloning of hybridoma cells

Screening positive holes by an ELISA detection method, and laying recombinant expression CD2v protein overnight; washing the plate, adding skimmed milk powder, sealing, and standing at 37 deg.C for 1 hr; washing the plate, adding 100 mul of culture solution supernatant of 96 holes, and incubating for 1h at 37 ℃; washing the plate, adding a goat anti-mouse secondary antibody marked by HRP, and incubating for 30min at 37 ℃; washing the plate, adding a developing solution, developing for 10min, adding a stop solution, and reading the numerical value of OD 450; screening high expression cell strain for subcloning culture.

4. Sequencing

The cells were collected, total RNA was extracted using Trizol, and cDNA was generated by reverse transcription using oligo (dT) 20 as a primer. Then, specific primers are used for PCR amplification of the heavy chain variable region gene and the light chain variable region gene respectively. And (3) after the PCR product is subjected to electrophoretic purification, inserting the PCR product into a vector through TA cloning, converting, and selecting positive clones for sequencing.

5D8 clones were screened and the sequencing results were as follows:

example 2, monoclonalDetection of physicochemical Properties of antibody 5D8

And (3) expressing and purifying the monoclonal antibody, chemically synthesizing the screened sequence, and cloning the sequence into a eukaryotic expression vector. And (5) amplifying and extracting the plasmid. Plasmids encoding the antibodies were transiently transfected into mammalian cells HEK293. Collecting the supernatant, and purifying by affinity chromatography to obtain the monoclonal antibody.

The expression level of the purified antibody was 142mg/L.

1. Gel electrophoresis for detecting purity of monoclonal antibody

1) Instrumentation and equipment

2) Primary reagent

Name (R)	Manufacturer of the product	Specification of	Goods number
				1M Tris-HCl buffer	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	60 ml/bottle	20200911
1.5M Tris-HCl buffer solution	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	100 ml/bottle	20200911
				10％SDS	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	10 ml/bottle	20200911
FastStain	Gene Universal	1000 ml/bottle	21DA
				30% glue solution (29	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	500 ml/bottle	20210414
Rainbow 180 broad-spectrum protein Marker	BEIJING SOLARBIO TECHNOLOGY Co.,Ltd.	250μl(50T)	1202F021

3) Sample preparation

Mixing 20 μ l sample with 5 μ l 5 × reducing buffer, heating at 95 deg.C for 5min, and cooling; mu.l of the sample was mixed well with 5. Mu.l of 5 Xnon-reducing buffer.

4) Electrophoresis

Preparing gel, adding a proper amount of electrophoresis buffer solution, adding sample, and performing electrophoresis.

5) Dyeing and bleaching

After electrophoresis, putting the gel into a proper amount of Coomassie brilliant blue staining solution, and staining for 1 hour or more at room temperature; pouring out the staining solution, adding appropriate amount of Coomassie brilliant blue staining decolorization solution, and decolorizing at room temperature for 4-24h. After completion of decolorization, ddH was used ₂ O soaking, comparing with the undyed gel according to Marker protein, cutting off the gel of the needed protein component, and collecting. The protein to be purified is then separated from the gel.

6) Results

The results are shown in FIG. 1, the bands from left to right are marker and reduction band; the detection purity of the monoclonal antibody is more than 95 percent.

2. HPLC detection of monoclonal antibody purity

1) Instrumentation and equipment

2) Primary reagent

Name (R)	Manufacturer of the product	Specification of	Goods number
				Dipotassium hydrogen phosphate trihydrate	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10017592
Potassium dihydrogen phosphate	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10017692
				Potassium chloride	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10016392

3) Fluid phase system

Dipotassium phosphate trihydrate, potassium dihydrogen phosphate and potassium chloride are added into about 900ml of purified water, stirred and dissolved, the volume is adjusted to 1L, and the pH value is determined to be between 6.2 +/-0.1 by measuring with a pH meter. Filtering with 0.22 μm filter membrane, and storing at room temperature.

4) Sample preparation

System applicability sample: MIL62 Standard substance diluted to 2mg/ml with mobile phase

And (3) testing the sample: the sample to be tested was diluted to 2mg/ml with mobile phase.

5) Chromatographic conditions

6) Results

As shown in FIG. 2, the purity of monoclonal antibody was greater than 95%.

Example 3 detection of binding Activity of monoclonal antibodies

1. Experimental procedure

1) Coating: diluting the antigen CD2v protein to 2 mu g/ml by using a coating solution, mixing uniformly, adding a 96-well coated plate, sealing by a membrane, sealing and keeping at 4 ℃ overnight.

2) The plate washer washes 3 times, no liquid can remain on the plate in the last time, and the liquid on the surface of the plate is patted dry by using absorbent paper.

3) And (3) sealing: add 5% milk powder (0.5 g milk powder in 10ml DPBS), 300. Mu.l/well, incubate 1h at 37 ℃ and wash plate 3 times as per step 2).

4) The antibody was diluted in a gradient of 100. Mu.l/well, reacted at 37 ℃ for 1h, and the plate was washed 3 times according to step 2).

5) Adding a secondary antibody: diluted with DPBS according to 1.

6) Color development: TMB was added at 100. Mu.l/well and developed in the dark at room temperature for 10min.

7) And (4) terminating: 2N H was added ₂

SO

₄ 100. Mu.l/well.

8) Measuring OD 450 with enzyme labeling instrument, and detecting within 10min.

2. As a result, the

The results are shown in FIG. 3, monoclonal antibody 5D8 can specifically bind to the antigen CD2v protein and exhibits a concentration dependence with an EC50 of 0.02535. Mu.g/ml.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof comprising light chain CDRs 1-3 and heavy chain CDRs 1-3,

the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.5, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.6, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 7;

preferably, the antibody includes monoclonal, chimeric, humanized, fully human and bispecific antibodies;

preferably, the antigen binding fragment comprises a Fab fragment, a Fab' fragment, a F (ab) ₂ Fragments, fv fragments and ScFv;

preferably, the CD2v comprises wild-type CD2v or a mutant thereof.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region of said antibody is represented by SED ID No. 8.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the variable region of the light chain of said antibody is represented by SED ID No. 9.

4. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of claim 1;

preferably, the nucleic acid molecule comprises DNA, RNA or a hybrid;

preferably, the nucleic acid molecule is double-stranded or single-stranded.

5. A vector comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of claim 1;

preferably, the vector is an expression vector;

preferably, the vector comprises a bacterial plasmid vector, a bacteriophage vector, a yeast plasmid vector, an adenoviral vector, a retroviral vector, or a lentiviral vector;

preferably, the expression vector further comprises a promoter and a transcription termination sequence operably linked thereto.

6. A cell comprising one or more of the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid molecule of claim 4, and the vector of claim 5;

preferably, the cells include prokaryotic cells, lower eukaryotic cells, higher eukaryotic cells;

preferably, the higher eukaryotic cells include animal cells;

preferably, the animal comprises a mouse, rat, hamster, pig, cow, horse, primate;

preferably, the primate is a human;

preferably, the cells include CHO cells, 293 cells, CHO-K1 cells, HEK293 cells, caco2 cells, U2-OS cells, NIH 3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, jurkat cells, hepG2 cells, heLa cells, HT-1080 cells, HCT-116 cells, hu-h7 cells, huvec cells, molt 4 cells.

7. A kit for detecting CD2v protein, said kit comprising any one or more of the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid molecule according to claim 4, the vector according to claim 5, and the host cell according to claim 6.

8. Use of any one of the antibody or antigen-binding fragment thereof according to claim 1, the nucleic acid molecule according to claim 4, the vector according to claim 5, the host cell according to claim 6, and the kit according to claim 7 for the detection of CD2v protein.

9. A method of producing the antibody or antigen-binding fragment thereof of claim 1, the method comprising expressing a nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1;

preferably, the method comprises culturing a cell comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1 in an environment that allows expression of the nucleic acid;

preferably, the method further comprises the steps of collecting and purifying the protein.

10. A method for detecting CD2v, the method comprising contacting a substance to be detected with any one or more of the antibody or antigen-binding fragment thereof of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6;

preferably, the test substance comprises a sample which may be from an animal;

preferably, the animals include cattle, sheep, pigs, horses, donkeys;

preferably, the sample comprises a nasal swab, purulent nasal fluid, genital swab, semen, respiratory mucosa, part of tonsil, lung, bronchial lymph node, liver, lung, kidney, and placental cotyledon;

preferably, the test object further comprises an environmental sample derived from a farm;

preferably, the environmental sample comprises air, water, soil, facilities of a farm;

preferably, the method may be combined with any one of: enzyme-linked immunosorbent assay, chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay and immunochromatography.