CN116970074A

CN116970074A - Monoclonal antibody combined with CD2v intracellular protein of African swine fever virus and application thereof

Info

Publication number: CN116970074A
Application number: CN202311185102.0A
Authority: CN
Inventors: 孙爱军; 庄国庆; 靳家鑫; 杜永坤; 张改平
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2022-09-15
Filing date: 2023-09-14
Publication date: 2023-10-31
Also published as: CN115368443A

Abstract

The invention provides a monoclonal antibody combined with an intracellular protein of a CD2v of an African swine fever virus and application thereof, belonging to the technical field of molecular immunization. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 3. The monoclonal antibody prepared by the invention can be specifically combined with the linear epitope of the CD2v intracellular region protein B cell, and has good reactivities. The monoclonal antibody recognizes the epitope to be highly conserved between different strains and different genotype strains, the homology reaches 100%, and the monoclonal antibody is helpful for further researching the molecular mechanism of the CD2v involved in the pathogenesis and immunity of ASFV and developing gene deletion vaccine.

Description

Monoclonal antibody combined with CD2v intracellular protein of African swine fever virus and application thereof

Technical Field

The invention relates to the technical field of molecular immunity, in particular to a monoclonal antibody combined with an intracellular protein of CD2v of African swine fever virus and application thereof.

Background

African Swine fever virus (African Swine FeverVirus, ASFV) infects pigs and causes African Swine fever (African Swine Fever, ASF) disease characterized by bleeding and fever. The world animal health Organization (OIE) lists it as legal report epidemic disease. ASFV mainly infects domestic pigs, wild pigs, and also can infect soft ticks. The infection of the high-pathogenicity strain can cause death of pigs for 4 to 15 days, and the death rate can reach 100 percent; subacute symptoms caused by moderate virulent strains have lower mortality (30-70%), most of which are hemorrhagic symptoms; and the low virulent strain has lower death rate, so that the chronic disease without vascular injury is caused.

ASFV belongs to the family African swine fever virus and genus African swine fever virus. The double stranded DNA genome is between 170 and 194kb in length, and is caused by the acquisition or deletion of portions of genes from different members of the multigene family (MGF), comprising 150 open reading frames, encoding about 200 proteins, including 68 structural proteins and over 100 non-structural proteins. Morphologically, ASFV virus particles are a symmetrical icosahedral particle, approximately 260nm in diameter, with a complex multi-enveloped structure, core-like, core-shell, internal lipid envelope, icosahedral capsid and outer envelope, respectively. The EP402R gene encodes a protein similar to the T cell adhesion molecule CD2, designated CD2v. CD2v comprises a glycoprotein assembled from a signal peptide, extracellular 2 immunoglobulin-like domains, a transmembrane region, and a C-terminal 147 amino acid intracellular domain, wherein the intracellular domain comprises 1 acidic domain and 1 proline-rich repeat. The intracellular domain of CD2v has no apparent identity to the amino acid sequence of the cytoplasmic domain of cellular CD2, and furthermore, confocal microscopy results indicate that the expressed CD2v protein structure is mostly located within the cell rather than on the cell surface. It has been found that CD2v interacts with SH3P7 through the proline repeat region, possibly modulating protein transport to participate in immunomodulation. The prior art studies found that AP-1 binds to the carboxy terminus of CD2v (230-304 aa) to form a CD2v-AP-1 complex, which has an effect on ASFV virulence and immune escape. Therefore, ASFV CD2v protein is an important target for development of vaccines and related diagnostic reagents. However, many studies in the prior art have been conducted on extracellular regions and full-length CD2v proteins, and polyclonal and monoclonal antibodies have been prepared, but there is a lack of research on intracellular regions of proteins.

Disclosure of Invention

The invention aims to provide a monoclonal antibody combined with an intracellular protein of a CD2v of an African swine fever virus and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a monoclonal antibody, the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.2, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 3.

Preferably, the monoclonal antibody specifically binds to a linear epitope of the B cell intracellular domain protein of african swine fever virus CD2v.

Preferably, the amino acid sequence of the linear epitope of the B cell of the intracellular region protein of the African swine fever virus CD2v is shown as SEQ ID NO. 1.

The invention also provides a nucleic acid molecule for encoding the monoclonal antibody, wherein the nucleic acid sequence for encoding the heavy chain variable region is shown as SEQ ID NO.4, and the nucleic acid sequence for encoding the light chain variable region is shown as SEQ ID NO. 5.

The invention also provides an expression vector comprising the nucleic acid molecule.

The invention also provides a host cell comprising the expression vector.

The invention also provides a pharmaceutical composition comprising the monoclonal antibody.

The invention also provides a detection reagent or a kit comprising the monoclonal antibody.

By adopting the technical scheme, the invention has the following beneficial effects: the invention provides a monoclonal antibody, the amino acid sequence of a heavy chain variable region of which is shown as SEQ ID NO.2, and the amino acid sequence of a light chain variable region of which is shown as SEQ ID NO. 3. The monoclonal antibody prepared by the invention can be specifically combined with the linear epitope of the CD2v intracellular region protein B cell, and has good reactivities. The monoclonal antibody recognizes the epitope to be highly conserved between different strains and different genotype strains, the homology reaches 100%, and the monoclonal antibody is helpful for further researching the molecular mechanism of the CD2v involved in the pathogenesis and immunity of ASFV and developing gene deletion vaccine.

Drawings

FIG. 1 shows the results of nucleic acid gel electrophoresis identification after construction of pET28b-CD2v for double digestion; wherein, the M lane is DNA molecular mass standard, the 1 lane is pET28b empty plasmid, and the 2 lane is pET28b-CD2v double enzyme cut product;

FIG. 2 is a diagram showing SDS-PAGE results of precipitation after induced expression of CD2v intracellular domain proteins, wherein M is a protein molecular weight standard; 1 is uninduced cell lysis precipitate, 2-8 is 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5 mmol.L respectively ^-1 IPTG-induced cell lysis pellet;

FIG. 3 is a diagram showing SDS-PAGE results of supernatants after induced expression of CD2v intracellular domain proteins, wherein M is a protein molecular weight standard; 1 is the uninduced cell lysis supernatant, 2-8 are 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5 mmol.L, respectively ^-1 IPTG-induced cell lysis supernatant;

FIG. 4 is a chart showing coomassie brilliant blue staining of CD2v intracellular domain proteins after purification and concentration; wherein M is a protein molecular weight standard;

FIG. 5 is a schematic representation of purification of the concentrated CD2v intracellular domain protein using Anti-His-mAb; wherein M is a protein molecular weight standard;

FIG. 6 is a schematic of purification of concentrated CD2v intracellular domain proteins using ASFV positive serum validation; wherein M is a protein molecular weight standard, and 1 is CD2v intracellular domain protein;

FIG. 7 shows the purity of purified CD2v intracellular domain protein by HPLC;

FIG. 8 is a diagram showing the identification of full length CD2v protein expressed by baculovirus insect cell expression system by using 1F3 monoclonal antibody through Westernblotting; wherein M is a protein molecular weight standard; 1 is sf9 cell control, 2 is sf9 cells expressing full length CD2v protein;

FIG. 9 is a PAM cell lysis pattern of 1F3 monoclonal antibodies expressed by a Westernblotting identification baculovirus insect cell expression system with infected and uninfected ASFV; wherein M is a protein molecular weight standard, 1 is a PAM cell control, and 2 is a PAM cell infected with ASFV;

FIG. 10 is a schematic of the identification of insect cells not vaccinated with baculovirus by indirect Immunofluorescence (IFA) with the 1F3 monoclonal antibody;

FIG. 11 is a diagram showing the identification of full length CD2v protein expressed by baculovirus insect cell expression system by 1F3 monoclonal antibody through indirect Immunofluorescence (IFA);

FIG. 12 shows the results of antigen index analysis of amino acids of the CD2v intracellular domain protein;

FIG. 13 shows the truncated overlap expression of the intracellular domain of the CD2v intracellular domain protein and Western Blot identification in example 4;

FIG. 14 is a graph showing the precise localization of the 1F3 monoclonal antibody recognition epitope region by WesternBlot using Anti-GFP-mAb and 1F3 monoclonal antibody;

FIG. 15 is a diagram showing an analysis of conservation of amino acids in the intracellular domain of the CD2v intracellular domain protein in different ASFV strains [ AminoAcid substitutions (. Times.100) ];

FIG. 16 is a diagram showing an analysis of the conservation of the 1F3 monoclonal antibody recognition epitope among different ASFV strains.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The materials and solutions used in the examples of the invention are as follows:

1. material

The pET28b plasmid and BL21 competent cells were purchased from Beijing full gold biotechnology Co., ltd, SPF grade BALB/c mice were purchased from Beijing Vitolith Lihua laboratory animal technology Co., ltd, and SP2/0 cells were stored in the laboratory.

Nickel columns were purchased from GE company, plasmid extraction kit, liquid paraffin, freund's complete adjuvant, freund's incomplete adjuvant were purchased from Sigma company, his-HRP monoclonal antibody, HRP-labeled goat anti-mouse IgG, FITC-labeled goat anti-mouse IgG were all purchased from Abcam company, restriction enzymes NdeI and XhoI were purchased from NEB company, UK, T4DNA ligase, BCA protein concentration assay kit were purchased from Promega and ThermoFisher company, respectively, mouse monoclonal antibody subtype identification ELISA kit was purchased from Proteintech company, ASFV positive serum was purchased from China veterinary medicine inspection, SDS-PAGE kit, ECL color development solution was purchased from Yase biotechnology gift biological company, TMB color development solution was purchased from Solebao biological company, and inactivated PAM cell protein samples were fed by Harbin veterinary research institute.

2. Solution preparation

(1) PBS buffer: weighing 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolving in 800mL deionized water, regulating pH to 7.4 with HCl, adding water to 1L, sterilizing at 121deg.C under high pressure for 20min, and preserving at 4deg.C;

(2) PBST buffer: adding 500 mu L of Tween-20 into 1L of PBS after high pressure, uniformly mixing, and preserving at 4 ℃;

(3) Sealing liquid: weighing 50g of skimmed milk, adding into 100mLPBST buffer, dissolving, and storing at 4deg.C;

(4) Coating buffer solution: 8.58g of Na2CO3.10H2O and 5.8g of NaHCO3 are weighed and added into deionized water, the volume is fixed to 1L, the PH is regulated to 9.5,4 ℃ for preservation;

(5) IPTG: 2g of IPTG was dissolved in 8mL of ultrapure water, the volume was set to 10mL, and the solution was sterilized by filtration through a 0.22 μm filter. Subpackaging into 1.5mL EP tubes, -20 for preservation;

(6) Kanamycin: dissolving 100mg kanamycin in enough ultrapure water, fixing the volume to 10mL, subpackaging into 1.5mL EP pipes, and preserving at-20 ℃;

(7) LB medium: weighing 10g of tryptone, 5g of yeast extract and 5g of NaCl, dissolving in 800mL of deionized water, regulating the pH to 7.4 by using NaOH, fixing the volume to 1L, sterilizing at 121 ℃ for 20min under high pressure, and preserving at room temperature for later use;

(8) Lysis buffer: 20mM Tris-HCl,150mM NaCl,pH 8.5;

(9) DMEM at 10% serum concentration: 50mL of FBS is added into 450mL of DMEM to prepare a DMEM cell culture solution of 10% FBS;

(10) Antibody neutralization buffer: 2mM bicine, 100mM KCl, 5mM MgCl2, 40. Mu.M cresol red (pH 8.0).

EXAMPLE 1 monoclonal antibody preparation

1. Construction of recombinant plasmid and purification and identification of protein expression

According to the ASFV Pic/HLJ/2018 strain published by NCBI Gene bank (GenBank ID: MK 333180.1) EP402R Gene sequence, a base fragment encoding 230-360 amino acids of the intracellular region of CD2v protein was optimized and synthesized by the Shake Marine engineering, ndeI and XhoI cleavage sites were introduced upstream and downstream of the fragment, the fragment was ligated with T4DNA ligase onto NdeI and XhoI double-digested pET28b expression vector (with GS linker and 6. Times. His tag added to the N-terminus), transformation was performed using TOP10 competent cells, single colonies were inoculated into LB liquid medium, placed on shaking table, cultured overnight at 37℃and subjected to cleavage verification. The positive plasmid identified by PCR was sent to Shanghai qingke company for sequencing. The recombinant plasmid with correct sequencing was named pET28b-EP402R, transformed into BL21 (DE 3) competent cells, the positive colony after transformation was cultured in LB medium of 30. Mu.g/ml kanamycin, when OD600 reached 0.5-0.6, at 25℃IPTG (isopropyl-. Beta. -d-thiogalactopyranoside) solution was added to a final concentration of 0.2mM.

After induction for 16h, bacterial pellet was collected by centrifugation, resuspended in lysis buffer (20 mM Tris-HCl,150mM NaCl,pH8.5), broken down for 15min at 4℃using a low temperature high pressure homogenizer, centrifuged for 60min at 4℃at 20,000Xg, cell debris removed, his affinity purified using a nickel column, recombinant CD2v intracellular domain protein eluted using 300mM imidazole, gel chromatography purified using HiLoad16/600Superdex 200pg and purity of the twice purified proteins detected by SDS-PAGE and HPLC. Finally, his-Tag monoclonal antibodies and African swine fever positive serum are used for respectively identifying CD2v intracellular region proteins.

After the synthesized CD2v truncated gene fragment is connected with the pET28b vector, ndeI and XhoI double-restriction enzyme digestion identification is carried out on pET28b-EP402R (figure 1), the strip size is correct, and the sequencing result of positive plasmid is consistent with that expected, thus indicating that the pET28b-EP402R prokaryotic expression vector is successfully constructed.

The non-induced groups, namely eight IPTG concentration induced groups of 0.1, 0.2, 0.4, 0.6, 0.8, 1 and 1.5mM, were respectively arranged, and nine temperature and time groups of 16 ℃ 8h, 16 ℃ 16h, 16 ℃ 24h, 25 ℃ 8h, 25 ℃ 12h, 25 ℃ 16h, 37 ℃ 4h, 37 ℃ 8h and 37 ℃ 12h were simultaneously arranged by 0.1mM IPTG concentration induction, and the maximum expression level of CD2v intracellular region protein under the conditions of 25 ℃ and 0.2mM IPTG induction 16h was determined after SDS-PAGE detection (FIGS. 2-3)

After a large amount of induced expression, the protein was purified by affinity chromatography and gel filtration chromatography, and the purity of the protein was detected to be 90% or higher by SDS-PAGE and HPLC (FIG. 4 and FIG. 7).

The purified CD2v intracellular domain protein is transferred onto PVDF membrane through Western Blotting, after skim milk is blocked, his monoclonal antibody and ASFV positive serum are used for respectively incubating overnight, and then diluted HRP marked goat anti-mouse IgG and goat anti-pig IgG are used for incubating for 1h at room temperature. The film was photographed in an imager and the results are shown in fig. 5 and 6, respectively. As can be seen from fig. 5 and 6, the recombinant ASFV CD2v intracellular domain protein exhibited a single specific band at a molecular weight of 17kDa, indicating that the recombinant ASFV CD2v intracellular domain protein had a strong antigen specificity after purification.

2. Monoclonal antibody preparation

CD2v intracellular domain protein and Freund's complete adjuvant emulsified mixture were immunized to 4-6 week old female BALB/c mice at a primary immunization dose of 80 μg/mouse. After 14d of priming, the emulsified mixture of CD2v intracellular domain protein and Freund's incomplete adjuvant was further immunized at a dose of 40. Mu.g/dose. According to the procedure of the second immunization, a third immunization is performed after 14 d. Blood is collected after 10d, serum is separated, the serum titer is detected by an indirect ELISA method, and a mouse with the highest serum titer is selected, and 100 mug of non-adjuvant CD2v intracellular domain protein is injected into the abdominal cavity for boosting. After spleen B cells of the boosted mice were isolated, cell fusion with SP2/0 cells was performed by a conventional method, and positive hybridoma cell selection was performed by an indirect ELISA method. Finally, through 2 subcloning, cell supernatant with the positive rate of 100% is obtained. Freezing and preserving after expansion culture, and preparing ascites by a conventional method.

The specific operation process of the indirect enzyme-linked immunosorbent assay is as follows:

96-well ELISA plates were coated with 100. Mu.L of recombinant protein at 5. Mu.g/ml, diluted with carbonate buffer (pH 9.6), incubated at room temperature for 2h, and blocked overnight with 300. Mu.L of 3% BSA at 4 ℃. After 3 times of washing of the blocked plates with PBST, 100. Mu.L of serum to be examined was added and incubated at 37℃for 1 hour. PBST was washed 3 times, sheep anti-mouse secondary antibody (1:5000 dilution) was labeled with 100. Mu. LHRP, incubated at 37℃for 30min, and washed 3 times with PBST. The reaction was stopped by adding 100. Mu.L of single-component substrate solution substrate/Kong Shiwen for 5min and then adding 50. Mu.L of 2M hydrochloric acid. Absorbance at a wavelength of 450nm was measured using a microplate reader, and the results were expressed as Optical Density (OD).

Results: after fusion with hybridoma cells, a monoclonal fusion cell line 1F3 is obtained through two rounds of subcloning screening.

3. Heavy chain and light chain variable region genes and amino acid sequences of monoclonal antibodies

The nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.4, the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.5, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 3.

EXAMPLE 2 monoclonal antibody identification

Westernblotting identification

The specificity of the 1F3 strain was measured by Westernblotting and reacted with baculovirus-expressed full-length CD2v protein (FIG. 8) and PAM cell lysates of infected and uninfected ASFV (FIG. 9), respectively.

The specific operation process of Westernblotting identification is as follows: baculovirus-expressed full-length CD2v protein and Sf9 cells and PAM cell lysates of infected and uninfected ASFV were electrophoretically separated on 12.5% SDS-PAGE gel, transferred to PVDF membrane, blocked with 5% skim milk at room temperature for 1h, followed by incubation with 1F3 monoclonal antibody (1:500) and ASFV positive serum (1:1000) at room temperature for 1h, respectively, followed by washing with PBST 3 times for 10min each, each incubation with goat anti-mouse HRP at a dilution of 1:3000 and goat anti-pig HRP-labeled secondary antibody at a dilution of 1:5000. After incubation for 1h at room temperature, PBST was washed 3 times, then a hypersensitive ECL (Enhanced Chemiluminescent) chemiluminescent chromogenic reagent was added and luminescence was allowed to occur for 1-5min using an Amersham Imager680 to identify the protein of interest. The results produced specific bands that were further detected by IFA method.

IFA method detection

Designing primers by taking the synthesized genes as templates, wherein the primers are shown as SEQ ID NO.6 and SEQ ID NO. 7:

CD2v-F:5’-gatcggatccggATGATCATCCTGATCTTCC-3’，SEQ ID NO.6；

CD2v-R:5’-gatcaagcttttaGATGATGCGATCGACG-3’，SEQ ID NO.7；

the full-length fragment of the EP402R gene is obtained by PCR amplification. The PCR product and the pFastBacHTC vector are subjected to double digestion by BamHI and HindIII, then are connected with T4 ligase at 16 ℃ overnight, are transformed into TOP10 competence, are coated on an LB solid culture medium with ampicillin resistance, and positive colonies are picked up in a liquid LB culture medium with ampicillin resistance to extract plasmids. The successfully constructed plasmid is transformed into DH10Bac competence, coated on LB solid medium containing the carban, tetracyclic, gentamicin resistance and IPTG and X-Gal, positive colony is selected by twice blue-white spot screening and shaken, and the rod grain DNA is extracted. Prepare 2 x 10 ⁶ Cells were plated into T25 and after 20min 8. Mu.g of recombinant bacmid were transfected. After 4 passages full length CD2v protein expression was detected and cell supernatants were harvested and stored at-80 ℃. Cell supernatants containing baculovirus at 200. Mu.L/wellToxicity was into 6-well plates in which sf9 insect cells were cultured. After 72h the medium in the 6-well plate was discarded, fixed with 4% paraformaldehyde and washed 3 times with PBS. Permeabilization was performed with 0.1% Triton x-100 and washed 3 times with PBS. Blocking with 10% FBS at room temperature for 30min, and washing with PBS for 3 times. The monoclonal antibody was diluted 1:100 with PBS and incubated for 1h at 37℃in an incubator and washed 3 times with PBS. Alexa-Fluor-488 conjugated goat anti-mouse secondary antibody was diluted 1:500 with PBS, incubated 1h at 37℃and washed 3 times with PBS and observed under a fluorescence microscope.

The results are shown in FIGS. 10 and 11. FIG. 10 shows the results of IFA detection of empty vector transfected wells, as can be seen from FIG. 10, no fluorescence is present; FIG. 11 shows the results of IFA detection of wells of Sf9 insect cells transfected with baculovirus, and from FIG. 11, a fluorescent reaction appears, which indicates that the generated monoclonal antibody can bind to the full-length CD2v protein expressed in Sf9 insect cells, and the monoclonal antibody has good reaction specificity, and is named mAb-CD2v-1F3.

EXAMPLE 3 subtype identification of monoclonal antibodies

Antibodies were classified by detecting monoclonal cell culture supernatants using mouse monoclonal antibody isotype reagents (Yiqiao shenzhou technologies, inc., china). According to the kit instructions (catalog number: SEK 003), 100. Mu.L of the culture supernatant of the monoclonal cell line 1F3 was added to each well for detection. Color development is carried out by using TMB single-component substrate solution substrate, absorbance is measured at 450nm, and under the condition that positive control is established, a positive threshold value is obtained by adding 4 times variance to the negative control result.

The results are shown in Table 1, showing that mAb-CD2v-1F3 monoclonal antibody subtype belongs to IgG1.

TABLE 1 identification of monoclonal antibody subclasses

Clone	mAb-CD2v-1F3
		IgG1	1.5
IgG2a	0.03
		IgG2b	0.03
IgG2c	0.02
		IgG3	0.04
Blank	0.02

EXAMPLE 4 identification of monoclonal antibody recognition epitopes

To determine the epitope recognized by the 1F3 monoclonal antibody, we cut off the amino acid analysis of the intracellular region of the CD2v protein of ASFV into three sections of C1 (230-290 aa), C2 (270-336 aa) and C3 (300-360 aa) according to DNAstar protein software, and ligate the nucleotide sequences corresponding to the three sections of amino acids on pEGFP-N1 mammalian expression vectors, respectively, and transfect 293T cells and express. Westernblotting identification was performed using anti-GFP-tagged monoclonal antibody and 1F3 monoclonal antibody, and the identification result showed that the epitope region recognized by the monoclonal antibody was 230-290aa. Then deleting and truncating N-terminal and C-terminal amino acids of the A1 segment protein into six segments of C1-1, C1-2, C1-3, C1-4, C1-5 and C1-6, expressing and identifying by the method, and the result is shown in figure 13, and as can be seen from figure 13, 1F3 reacts with the designed six segments of truncated protein, and the position of the epitope at the overlapped part 262-275aa of C1-4 and C1-6 can be determined.

According to the epitope recognition result, the amino acid sequence corresponding to the peptide fragment in Table 2 was synthesized, ligated to pEGFP-N1 vector, 293T cells were transfected with pEGFP-N1 vector and expressed, and after the above method was used for fixation, the epitope amino acid sequence determined from FIG. 14 was shown as SEQ ID NO. 1.

TABLE 2 peptide amino acid sequences for epitope identification

SEQ ID NO.1：EPSPREP

Example 5CD2v intracellular region protein epitope conservation analysis

In order to analyze the conservation of the 1F3 monoclonal antibody recognition epitope in different african swine fever strains, multiple homology alignments were performed of the peg/HLJ/2018 strain with CD2v amino acid sequence information of other ASFV strains. The conservation of the monoclonal antibody recognition epitope in ASFV strains from different countries or regions was analyzed using Unipro UGENE software and the results are shown in fig. 16.

As can be seen from FIG. 16, the amino acid sequence of the CD2v intracellular region protein of the Pic/HLJ/2018 strain has 100% homology with the ASFV strain of the same genotype, 66.6% homology with the ASFV strain of different genotypes (FIG. 15), but the 1F3 monoclonal antibody recognition epitope is highly conserved between the different strains and the strains of different genotypes, and the homology reaches 100%.

As can be seen from the above examples, the invention optimizes the E.coli codon according to the gene sequence of the ASFV Pig/HLJ/2018 strain EP402R published by GenBank (GenBank ID: MK 333180.1), synthesizes the whole sequence of the optimized gene, intercepts the intracellular region fragment of EP402R, and successfully expresses the soluble recombinant protein pET28b-EP402R-His. The purified CD2v intracellular domain protein can be specifically combined with ASFV positive serum, so that the protein has good reactivities. The heavy chain subtype of the monoclonal antibody (18-2-A1-F3 strain) prepared by the monoclonal antibody is of an IgG1 type, can specifically bind to CD2v protein expressed in PAM cells infected with ASFV and also can bind to CD2v protein expressed in Sf9 insect cells, and shows that the prepared monoclonal antibody can possibly recognize CD2v conformational epitope, and is helpful for further researching the molecular mechanism of the CD2v participating in the pathogenesis and immunization of the ASFV and developing gene deletion vaccine.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

SEQ ID NO.1：

EPSPREP

SEQ ID NO.2:

QLQESGGGLVQPGGSMKLSCAASGFPFSDAWMDWVRQSPERGLEW VAEIRSKANNHATYYAESVKGRFTISRDDSKSRVYLHMNSLRGEDTGIYY CNGPAAGVWGQGTTVTVSS；

SEQ ID NO.3:

DIELTQSPVSITASRGEKVTITCRANSSISSNNFHWYLRRPGSSPKLLIYRTSILASGVLDSFSGRGSESSSTLTIHYMQDEVAATYYCQQGSICPPRSEGGPSSRSN

SEQ ID NO.4：

CAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCCCTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAGGGGGCTTGAGTGGGTTGCTGAAATTAGAAGCAAAGCTAATAATCATGCAACATACTATGCTGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTCGTGTCTACCTACATATGAACAGCTTAAGAGGTGAAGACACTGGCATTTATTACTGTAATGGTCCAGCTGCAGGAGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA；

SEQ ID NO.5：

GACATTGAGCTCACCCAGTCTCCAGTATCCATAACTGCATCTCGAGGGGAGAAGGTCACCATCACCTGCCGTGCCAACTCAAGTATAAGTTCCAATAACTTCCACTGGTACCTGCGGAGGCCAGGATCCTCCCCTAAACTTTTGATTTATAGAACATCCATCCTGGCATCTGGAGTCCTAGACAGCTTCAGTGGCCGTGGGTCTGAGAGTTCTTCCACTCTGACAATCCACTACATGCAGGACGAAGTTGCTGCCACTTACTATTGTCAGCAGGGGAGTATTTGTCCACCACGTTCGGAGGGGGGACCAAGCTCGAGATCTAAC。

Claims

1. A monoclonal antibody is characterized in that the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.2, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID NO. 3.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically binds to a linear epitope of B-cell, an intracellular domain protein of CD2v of african swine fever virus.

3. The monoclonal antibody according to claim 2, wherein the amino acid sequence of the linear epitope of the B cell of the intracellular domain protein of african swine fever virus CD2v is shown in SEQ ID No. 1.

4. A nucleic acid molecule encoding the monoclonal antibody of claim 1, wherein the nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID No.4 and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID No. 5.

5. An expression vector comprising the nucleic acid molecule of claim 3.

6. A host cell comprising the expression vector of claim 4.

7. A pharmaceutical composition comprising the monoclonal antibody of claim 3.

8. A detection reagent or kit comprising the monoclonal antibody of claim 1.