CN115368443A

CN115368443A - African swine fever virus CD2v protein B cell linear epitope and application

Info

Publication number: CN115368443A
Application number: CN202211129845.1A
Authority: CN
Inventors: 庄国庆; 孙爱军; 路闻龙; 张昂克; 张改平
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2022-09-15
Filing date: 2022-09-15
Publication date: 2022-11-22
Also published as: CN116970074A

Abstract

The invention discloses a linear epitope of B cells of CD2v protein of African swine fever virus, belonging to the technical field of biology. The amino acid sequence of the African swine fever virus CD2v protein B cell linear epitope is shown in SEQ ID NO. 1. The invention provides a B cell linear epitope of an African swine fever virus CD2v protein, a monoclonal antibody obtained by stimulation of the B cell linear epitope and application thereof, and provides new thinking for researching the intracellular region function of the CD2v protein and establishing a serological diagnosis method.

Description

African swine fever virus CD2v protein B cell linear epitope and application

Technical Field

The invention relates to the technical field of molecular immunity, in particular to a B cell linear epitope of an African swine fever virus CD2v protein, a monoclonal antibody obtained by utilizing the B cell linear epitope for stimulation, and application of the B cell linear epitope and application of the monoclonal antibody generated by the B cell linear epitope for stimulation.

Background

African Swine Fever Virus (ASFV) infects pigs, causing African Swine Fever (ASF) disease characterized primarily by bleeding and Fever. The world animal health Organization (OIE) lists it as a legal report epidemic. ASFV mainly infects domestic pigs and wild pigs, and can also infect ticks. The high-pathogenicity strain infection can cause the death of the pig for 4 to 15 days, and the disease death rate can reach 100 percent; subacute symptoms caused by mesogenic strains, the death rate is low (30-70%), and most of the hemorrhagic symptoms appear; and the low-toxicity strain has low mortality rate, resulting in chronic diseases without vascular injury.

ASFV belongs to African swine fever virus family, african swine fever virus genus. The double-strand DNA genome is 170-194 kb in length, is caused by the acquisition or deletion of partial genes of different members of a multigene family (MGF), comprises more than 150 open reading frames, and encodes about 200 proteins, including 68 structural proteins and more than 100 non-structural proteins. Morphologically, an ASFV virion is a symmetrical icosahedral particle with a diameter of about 260nm, having a complex multi-envelope structure, from inside to outside, a nucleoid, a nucleocapsid, an inner lipid envelope, an icosahedral capsid and an outer envelope. The EP402R gene encodes a protein similar to the T cell adhesion molecule CD2, referred to as CD2v. CD2v comprises a glycoprotein assembled from a signal peptide, an extracellular 2 immunoglobulin-like domain, a transmembrane region, and a C-terminal 147 aa intracellular domain, wherein the intracellular domain comprises 1 acidic domain and 1 proline-rich repeat. The intracellular domain of CD2v has no significant identity to the amino acid sequence of the cytoplasmic domain of cellular CD2, and in addition, confocal microscopy results indicate that the expressed CD2v protein structure is located mostly inside the cell rather than on the cell surface. It has been found that CD2v interacts with SH3P7 through the proline repeat region and may regulate protein trafficking to participate in immune regulation. Prior art researches find that AP-1 is combined with CD2v carboxyl terminal (230-304 aa) to form a CD2v-AP-1 complex, and the CD2v-AP-1 complex has an influence on ASFV virulence and immune escape. Therefore, the ASFV CD2v protein is an important target point related to the development of vaccines and related diagnostic reagents. However, most of the studies in the prior art are extracellular domain and full-length CD2v protein, and polyclonal and monoclonal antibodies are prepared, while the studies on intracellular domain of the protein are lacked.

Disclosure of Invention

The invention provides a CD2v protein monoclonal antibody against African swine fever virus and application thereof aiming at researching a small number of CD2v protein intracellular regions, screens out a B cell linear epitope specifically identified by the antibody, and provides new thinking for researching CD2v protein intracellular region functions and establishing a serological diagnosis method.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a linear epitope of B cells of CD2v protein of African swine fever virus, and the amino acid sequence of the linear epitope is shown in SEQ ID NO. 1.

In a second aspect of the invention, the application of the African swine fever virus CD2v protein B cell linear epitope in the preparation of a reagent for detecting or preventing African swine fever virus is provided.

In a third aspect of the invention, a monoclonal antibody against African swine fever virus CD2v protein is provided, wherein the monoclonal antibody can be specifically combined with a B cell linear epitope of the African swine fever virus CD2v protein.

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

In a fourth aspect of the invention, there is provided a nucleic acid molecule encoding said monoclonal antibody.

Preferably, the nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO.2 and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO. 3.

In a fifth aspect of the invention, there is provided an expression vector comprising said nucleic acid molecule.

In a sixth aspect of the invention, there is provided a host cell comprising said expression vector.

In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising said monoclonal antibody.

In an eighth aspect of the invention, a detection reagent or a kit comprising the monoclonal antibody is provided.

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

the invention obtains a monoclonal antibody cell strain which can identify epitope peptide information as shown in SEQ ID NO.1 by preparing a mouse monoclonal antibody, utilizing western blotting, indirect immunofluorescence IFA and enzyme-linked immunosorbent assay, and specifically identifies the monoclonal antibody of ASFV CD2v, thereby having the application potential of developing ASFV vaccine and diagnosis method in the next step.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the result of nucleic acid gel electrophoresis identification after double digestion of pET28b-CD2 v; wherein, the lane M is a DNA molecular mass standard, the lane 1 is a pET28b no-load plasmid, and the lane 2 is a product obtained by double enzyme digestion of pET28b-CD2 v;

FIG. 2 is a SDS-PAGE result of precipitation after induced expression of CD2v protein, wherein M is a protein molecular weight standard; 1 is non-induced cell lysis precipitate, and 2-8 are respectively 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5 mmol.L ^-1 IPTG-induced cell lysis precipitation;

FIG. 3 is a photograph showing the results of SDS-PAGE of the supernatant after induced expression of CD2v protein, wherein M is a protein molecular weight standard; 1 is the uninduced cell lysis supernatant, and 2-8 are 0.1, 0.2, 0.4, 0.6, 0.8, 1, respectively.5mmol·L ^-1 IPTG-induced cell lysis supernatant;

FIG. 4 is a photograph of purified and concentrated CD2v protein stained with Coomassie Brilliant blue; wherein M is a protein molecular weight standard;

FIG. 5 is a validation of purified concentrated CD2v protein using Anti-His-mAb; wherein M is a protein molecular weight standard;

figure 6 is a validation of purified concentrated CD2v protein using ASFV positive serum; wherein M is a protein molecular weight standard, and 1 is a CD2v protein;

FIG. 7 shows the high performance liquid chromatography for the purity of purified CD2v protein;

FIG. 8 shows that the 1F3 monoclonal antibody identifies the full-length CD2v protein expressed by the baculovirus insect cell expression system by Western blotting; wherein M is a protein molecular weight standard; 1 is sf9 cell control, 2 is sf9 cells expressing full length CD2v protein;

FIG. 9 shows that the 1F3 monoclonal antibody identifies PAM cell lysis samples of infected and uninfected ASFV expressed by baculovirus insect cell expression system by Western blotting; 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 shows the identification of non-baculovirus inoculated insect cells by the 1F3 monoclonal antibody by indirect Immunofluorescence (IFA);

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

FIG. 12 is an analysis of the antigenic index of amino acids of the CD2v protein;

FIG. 13 is a truncated and overlapping expression of the intracellular domain of CD2v protein according to FIG. 12 and identification of truncated and overlapping expressed recombinant CD2v protein by Western Blot using Anti-GFP-mAb and 1F3 monoclonal antibody; A. b is the truncated overlapping expression of the intracellular region of the CD2v protein; c is 1F3 monoclonal antibody and anti-gfp antibody to detect A truncated and overlapped expressed protein; d is 1F3 monoclonal antibody and anti-gfp antibody to detect B truncated and overlapped expressed protein;

FIG. 14 is a diagram showing the precise localization of the epitope region recognized by the 1F3 monoclonal antibody by Western Blot using Anti-GFP-mAb and the 1F3 monoclonal antibody;

FIG. 15 is an analysis of the conservation of intracellular domain Amino acids of the CD2v protein among different strains of ASFV (Amino Acid substitutions (. Times.100));

FIG. 16 is a conservative analysis of the recognition epitope of the 1F3 monoclonal antibody among different ASFV strains.

Detailed Description

The technical scheme of the invention is clearly and completely described in the following with reference to the accompanying drawings. It is to be understood that the described embodiments are only some of the embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

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

1. material

pET28b plasmid and BL21 competent cells were purchased from Beijing Quanjin Biotechnology, inc., SPF-grade BALB/c mice were purchased from Beijing Wittingli laboratory animal technology, inc., and SP2/0 cells were stored in the laboratory.

The nickel column was purchased from GE, the plasmid extraction kit, liquid paraffin, freund's complete adjuvant, freund's incomplete adjuvant was purchased from Sigma, his-HRP monoclonal antibody, HRP-labeled goat anti-mouse IgG, FITC-labeled goat anti-mouse IgG were purchased from Abcam, the restriction enzymes NdeI and XhoI were purchased from NEB, the T4DNA ligase, BCA protein concentration determination kits were purchased from Promega and ThermoFisher, respectively, the mouse monoclonal antibody subtype identification ELISA kit was purchased from Proteitech, the ASFV positive serum was purchased from China veterinary medicine monitoring institute, the SDS-PAGE kit, ECL scientific and chromogenic solution were purchased from Yazyme Biocolor development Limited, the TMB solution was purchased from Sorobao Biochemical, and the inactivated PAM cell protein sample was presented from Harbin veterinary institute.

2. Solution preparation

(1) PBS buffer: weighing 8.0g NaCl, 0.2g KCl and 1.44g Na ₂ HPO ₄ 、0.24g KH ₂ PO ₄ Dissolving in 800mL deionized water, adjusting pH to 7.4 with HCl, adding water to desired volume of 1L, autoclaving at 121 deg.C for 20min, and storing at 4 deg.C for preparationUsing;

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

(3) Sealing liquid: weighing 50g of skimmed milk, adding into 100mL of PBST buffer solution, dissolving, and storing at 4 ℃;

(4) Coating buffer solution: 8.58g of Na were weighed ₂ CO ₃ ·10H ₂ O and 5.8g NaHCO ₃ Adding into deionized water, diluting to 1L, adjusting pH to 9.5, and storing at 4 deg.C;

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

(6) Kanamycin: dissolving 100mg kanamycin in sufficient ultrapure water, diluting to 10mL, subpackaging into 1.5mL EP tubes, 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, adjusting the pH value to 7.4 by using NaOH, fixing the volume to 1L, carrying out autoclaving at 121 ℃ for 20min, and storing 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 was added to 450mL of DMEM to prepare a 10-vol% DMEM cell culture solution containing FBS;

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

Example 1

Preparation of monoclonal antibodies

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

According to the Gene sequence of EP402R of ASFV Pig/HLJ/2018 strain (GenBank ID: MK 333180.1) published by NCBI Gene bank, a truncated EP402R Gene is optimized in Shanghai life, ndeI and XhoI enzyme cutting sites are introduced at the upstream and the downstream, a base fragment coding 230-360 amino acids of an intracellular region of a CD2v protein is synthesized, the fragment is connected to a NdeI and XhoI double-cut pET28b expression vector (a GS linker and a 6 × His tag are added at the N end) by using T4DNA ligase, and TOP10 competent cells are used for enteringAnd (3) transforming, inoculating a single colony to an LB liquid culture medium, placing the colony on a shaking table, culturing at 37 ℃ overnight, extracting the plasmid, and performing enzyme digestion verification. The positive plasmids identified by PCR were sent to Shanghai Protechs company for sequencing. Named and sequenced correctly recombinant plasmid pET28b-EP402R, transformed into BL21 (DE 3) competent cells, the transformed positive colony, in 30 u g/ml kanamycin LB medium culture, when OD ₆₀₀ When the concentration reached 0.5-0.6, IPTG (isopropyl-. Beta. -d-thiogalactopyranoside) solution was added to a final concentration of 0.2mM at 25 ℃.

After 16h of induction, the pellet was collected by centrifugation, resuspended in lysis buffer (20 mM Tris-HCl,150mM NaCl, pH 8.5), disrupted for 15min at 4 ℃ using a low temperature high pressure homogenizer, the lysate was centrifuged for 60min at 4 ℃ and 20,000 Xg to remove cell debris, his affinity purification was performed using a nickel column, recombinant CD2v protein was eluted using 300mM imidazole, the eluted CD2v protein was purified by gel chromatography using HiLoad16/600Superdex 200pg, and the purity of the twice purified protein was checked by SDS-PAGE and HPLC. Finally, the truncated CD2v protein is respectively identified by using a His-Tag monoclonal antibody and African swine fever positive serum.

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

By optimizing the expression conditions, it was confirmed that the expression level of CD2v protein was the greatest under the condition of inducing with 0.2mM IPTG at 25 ℃ for 16h (FIG. 2-3).

After a large amount of induction expression, the protein is purified by affinity chromatography and gel filtration chromatography, and the purity of the protein is detected to be more than 90 percent by SDS-PAGE and high performance liquid chromatography (figure 4 and figure 7).

The purified CD2v protein is detected by using a His monoclonal antibody (figure 5) and ASFV positive serum (figure 6) Western Blotting, and the result shows that the recombinant ASFV CD2v protein has good antigenicity.

2. Preparation of monoclonal antibodies

A4-6 week-old female BALB/c mouse is immunized with the emulsified mixture of CD2v protein and Freund's complete adjuvant, and the primary immunization dose is 80 mu g/mouse. After the initial immunization for 14 days, the emulsified mixture of CD2v protein and Freund's incomplete adjuvant was re-immunized at a dose of 40. Mu.g/mouse. Following the protocol of the second immunization, a third immunization was performed after 14 d. After 10 days, blood is collected and serum is separated, the indirect ELISA method is used for detecting the serum titer, the mouse with the highest serum titer is selected, and 100 mu g of CD2v protein without adjuvant is injected into the abdominal cavity for boosting immunity. After spleen B cells of the booster-immunized mice were isolated, cell fusion was performed with SP2/0 cells by a conventional method, and positive hybridoma screening was performed by an indirect ELISA method. Finally, cell supernatants with 100% positive rate were obtained by 2 subcloning. Freezing and storing after expanding 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 plate coating 100 μ L of 5 μ g/ml recombinant protein was diluted with carbonate buffer (pH 9.6), incubated at room temperature for 2h, blocked overnight with 300 μ L of 3% BSA at 4 ℃. The sealed plate is washed 3 times with PBST, then 100. Mu.L of serum to be detected is added, and incubation is carried out for 1h at 37 ℃. PBST was washed 3 times, with 100 μ LHRP-labeled goat anti-mouse secondary antibody (1: 5000 dilution), incubated at 37 ℃ for 30min, and washed 3 times with PBST. 100 μ L of TMB monocomponent substrate solution substrate/well was added and incubated for 5min, and then 50 μ L of 2M hydrochloric acid was added to stop the reaction. Absorbance at a wavelength of 450nm was measured using a microplate reader and the results were expressed in terms of Optical Density (OD).

As a result: after being fused with hybridoma cells, a monoclonal fused cell line 1F3 is obtained through two rounds of subclone screening.

3. Heavy chain and light chain variable region gene and amino acid sequence of monoclonal antibody

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

Example 2

Monoclonal antibody identification

The reaction specificity of the 1F3 antibody was examined by Western blotting and reacted with the full-length CD2v protein expressed by baculovirus (FIG. 8) and the PAM cell lysate infected with and not infected with ASFV (FIG. 9), respectively.

The specific operation process of Western blotting identification is as follows: the recombinant proteins were electrophoretically separated in a 12.5% SDS-PAGE gel, the proteins were transferred to PVDF membrane, blocked with 5% skim milk at room temperature for 1h, then incubated with 1F3 monoclonal antibody (1: 500) and ASFV positive serum (1: 1000), respectively, at room temperature for 1h, then washed 3 times with PBST, 10min each, and then incubated 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, respectively. After incubation for 1h at room temperature, PBST was washed 3 times, followed by addition of hypersensitivity ECL (Enhanced chemiluminiscence) Chemiluminescent chromogenic reagent and luminescence for 1-5min using Amersham Imager 680, to identify the protein of interest.

All results produced specific bands, which were further detected by IFA method. The IFA detection comprises the following specific operation steps:

designing a primer by taking the synthesized gene as a template:

CD2 v-F5 '-galtcggatcggATGATCATCCTGATCTTCC-3' as shown in SEQ ID NO.6, CD2 v-R5 '-galtcaagcttttaGATGATGCGACGATCGACG-3' as shown in SEQ ID NO. 7;

the full-length fragment of the EP402R gene was obtained by PCR amplification. The PCR product and the pFastBacHTC vector are subjected to double enzyme digestion through BamHI and HindIII, then are connected overnight at 16 ℃ by T4 ligase, and are transformed into TOP10 competence, coated on an aminobenzyl-resistant LB solid culture medium, and a positive colony is selected in an aminobenzyl-resistant liquid LB culture medium to extract a plasmid. The successfully constructed plasmid is transformed into a DH10Bac competence, is coated on an LB solid culture medium containing kanamycin, tetracyclo, gentamicin resistance, IPTG and X-Gal, and positive bacterial colonies are selected and shaken by two times of blue-white screening to extract bacmid DNA. Preparation 2X 10 ⁶ Cells were plated in T25 and 20min later 8. Mu.g of recombinant bacmids were transfected. Full-length CD2v protein expression was detected after 4 passages, cell supernatants were harvested and stored at-80 ℃. 200 μ L/well of cell supernatant containing baculovirus was inoculated into 6-well plates in which sf9 cells were cultured. After 72h, the medium in the 6-well plate was discarded, fixed with 4% paraformaldehyde, and washed with PBS3 times. Then, the cells were permeabilized with 0.1% Triton x-100 and washed 3 times with PBS. Blocking with 10% FBS at room temperature 30min, washing with PBS 3 times. The monoclonal antibody was diluted 1: 100 with PBS, incubated at 37 ℃ in an incubator for 1h and washed 3 times with PBS. Alexa-Fluor-488 conjugated goat anti-mouse secondary antibody was diluted 1: 500 with PBS, incubated at 37 ℃ for 1h, washed 3 times with PBS, and observed under a fluorescence microscope.

The results show that this monoclonal antibody can bind to the full-length CD2v protein expressed in Sf9 cells (fig. 11), with strong specificity, and no fluorescence is present in the empty vector transfection well control (fig. 10).

The above results show that the monoclonal antibody has good reaction specificity, and the monoclonal antibody is named as mAb-CD2v-1F3.

Example 3

Subtype identification of monoclonal antibodies

The antibodies were classified by assaying monoclonal cell culture supernatants using mouse monoclonal antibody isotyping reagents (Yinqiao Shenzhou science Co., ltd., china). According to the kit instructions (catalog No. SEK 003), 100. Mu.L of the culture supernatant was added to each well to carry out the assay. Developing with TMB single-component substrate solution substrate, measuring absorbance at 450nm, and adding 4 times of variance to the negative control result as positive threshold of the determination result under the condition that the positive control is established.

The results showed that the subtype of the 1F3 monoclonal antibody was IgG1 (Table 1).

TABLE 1 identification of monoclonal antibody subclasses

Clone	18-2-A1-F3
		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 epitope

In order to determine the epitope recognized by the 1F3 monoclonal antibody, the amino acid analysis of the CD2v protein intracellular region of ASFV is cut into three sections of A1 (230-290 aa), A2 (270-336 aa) and A3 (300-360 aa) according to DNAstar Protean software, corresponding base sequences are connected on a pEGFP-N1 vector and transfected into 293T cells for expression, western blotting identification is carried out by using an anti-GFP label monoclonal antibody and the 1F3 monoclonal antibody, and the epitope region recognized by the monoclonal antibody is 230-290aa. And the A1 segment of protein is truncated into six segments by deleting the amino acids at the N end and the C end, and after the expression and the identification by the method, the epitope is further determined to be A1-6 (262-275 aa) (FIG. 12). The corresponding gene sequence of the peptide fragment (Table 2) was synthesized based on the epitope recognition result, and the pEGFP-N1 vector was ligated, and the amino acid sequence for precisely locating the epitope region by the above method was shown as SEQ ID NO.1 (FIGS. 13 to 14).

TABLE 2 peptide fragment amino acid sequences for epitope identification

Amino acid position	Amino acid sequence
		CD2v-262-271	IHEPSPREPL
CD2v-262-270	IHEPSPREP
		CD2v-263-271	HEPSPREPL
CD2v-264-270	EPSPREP

Example 5

CD2v protein intracellular region epitope conservation analysis

In order to analyze the conservation of the recognition epitope of the 1F3 monoclonal antibody in different African swine fever strains, the CD2v amino acid sequence information of the Pig/HLJ/2018 strain and other ASFV strains are subjected to multiple homologous alignment. The conservation of the monoclonal antibody recognizing epitopes in ASFV strains from different countries or regions was analyzed by Unipro UGENE software.

The results showed that the CD2v protein amino acid sequence of the Pig/HLJ/2018 strain had 100% homology with the ASFV strain of the same genotype and 66.6% homology with the ASFV strain of different genotypes (fig. 15), but the epitope recognized by the 1F3 monoclonal antibody was highly conserved between different strains and different genotype strains and had 100% homology (fig. 16).

According to the invention, escherichia coli codon optimization is carried out according to a Chinese isolated ASFV Pig/HLJ/2018 strain EP402R gene sequence (GenBank ID: MK 333180.1) published by GenBank, the optimized gene is subjected to complete sequence synthesis, an intracellular region fragment of EP402R is intercepted, and a soluble recombinant protein pET28b-EP402R-His is successfully expressed. The purified intracellular CD2v protein can be specifically combined with ASFV positive serum, which indicates that the protein has good reactogenicity. The heavy chain subtype of the monoclonal antibody (18-2-A1-F3 strain) prepared by the monoclonal antibody is IgG1 type, can be specifically combined with CD2v protein expressed in PAM cells infected with ASFV, and can also be combined with CD2v protein expressed in Sf9 cells, so that the prepared monoclonal antibody can possibly identify CD2v conformational epitope, and is helpful for further researching the molecular mechanism of CD2v participating in ASFV pathogenesis and immunity and developing gene deletion vaccines.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A B cell linear epitope of African swine fever virus CD2v protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. Use of the linear B-cell epitope of the African swine fever virus CD2v protein of claim 1 in preparation of a reagent for detecting or preventing African swine fever virus.

3. A monoclonal antibody against African swine fever virus CD2v protein, which is capable of specifically binding to the B cell linear epitope of African swine fever virus CD2v protein of claim 1.

4. The monoclonal antibody according to claim 3, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID No.4, and the amino acid sequence of the light chain variable region is represented by SEQ ID No. 5.

5. A nucleic acid molecule encoding the monoclonal antibody of claim 4.

6. The nucleic acid molecule of claim 5, wherein the nucleic acid sequence encoding the heavy chain variable region is set forth in SEQ ID No.2 and the nucleic acid sequence encoding the light chain variable region is set forth in SEQ ID No. 3.

7. An expression vector comprising the nucleic acid molecule of claim 5 or 6.

8. A host cell comprising the expression vector of claim 7.

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

10. A detection reagent or kit comprising the monoclonal antibody of claim 3.