CN115073609A

CN115073609A - Recombinant protein based on extracellular domain of African swine fever virus CD2v protein, and construction method and application thereof

Info

Publication number: CN115073609A
Application number: CN202210401397.XA
Authority: CN
Inventors: 杨毅; 邹亚文; 王乃东; 杨文兵; 王昌建
Original assignee: Hunan Paizhi Biotechnology Co ltd
Current assignee: Hunan Paizhi Biotechnology Co ltd
Priority date: 2022-04-18
Filing date: 2022-04-18
Publication date: 2022-09-20

Abstract

The invention discloses a recombinant protein based on an extracellular domain of African swine fever virus CD2v protein, which comprises the following components: the extracellular domain of the African swine fever virus CD2v protein is inserted with MBP tag protein at the N end of the extracellular domain of the African swine fever virus CD2v protein, or inserted with GCN4 motif or C-Jun motif at the C end of the extracellular domain of the African swine fever virus CD2v protein. Also discloses a construction method of the recombinant protein, application of the recombinant protein in establishing an indirect ELISA detection method, an indirect ELISA detection kit and a detection method. The recombinant protein can be subjected to soluble expression and purification in expression systems of mammals, baculovirus and escherichia coli, has the potential of distinguishing wild viruses from CD2v deletion strains, has serum specificity and distinguishing ASFV type 4 and type 8 serum types, can detect ASFV antibodies based on ELISA of CD2v extracellular domain, has higher sensitivity and specificity, and is widely applied.

Description

Recombinant protein based on extracellular domain of African swine fever virus CD2v protein and construction method and application thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to several recombinant proteins based on an extracellular domain of African swine fever virus CD2v protein, a construction method thereof, and application in establishing an indirect ELISA detection kit or an indirect ELISA detection method.

Background

African Swine Fever (ASF) is an acute infectious disease threatening the swine industry worldwide. African Swine Fever Virus (ASFV) is a double-stranded DNA (dsDNA) virus, with a genome size of 170-190 kbp, comprising 150 Open Reading Frames (ORFs). Mass spectrometry shows that at least 40 virus proteins are arranged from outside to inside in 5 virus structural domains of an outer envelope, a capsid, an inner envelope, a nucleocapsid and a nucleolus of the virus particles. There are 23 different genotypes of ASFV based on molecular genotyping of p72 (B646L). A simple serotyping method is based on the gene sequence of the CD2v (EP402R) or c-type lectin (EP153) proteins. CD2v and c-type lectin are necessary and sufficient conditions to mediate the serological specificity of the blood adsorption inhibition assay (HAI), and ASFV is currently divided into 8 serotypes according to HAI.

According to the molecular characteristics of p72 and CD2v, the pathogenic strain belongs to the serogroups of genotype II and 8, and has high virulence and infectivity in domestic pigs. In domestic pigs, the clinical symptoms of ASFV include high fever, bleeding, ataxia and major depression, with mortality approaching 100%. In the absence of a commercial ASFV vaccine, monitoring of ASFV is important in disease prevention and control.

Serological screening of a large number of clinical specimens for ASFV in Spain is considered a convenient and effective method for disease detection and eradication. The detection of ASFV antibodies is currently internationally performed mainly with three commercial kits (IDvet, Svanova and Ingenasa). ID vet is an indirect ELISA kit based on 3 recombinant proteins p32(p30), p72 and p 62; svanova is an indirect ELISA kit based on recombinant protein p 30; ingenasa is a competitive ELISA kit based on an anti-p 72 monoclonal antibody (mAb). Although all of these commercial kits can be used for detection of ASFV IgG, since ASFV serotypes and genotypes are numerous and spread rapidly, ASFV diagnostic kits having higher sensitivity and specificity and highly sensitive and widely used serological detection methods are urgently needed to control spread of the disease.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and defects in the background art and provide several recombinant proteins based on the extracellular domain of the African swine fever virus CD2v protein, a construction method and application thereof, an indirect ELISA kit and a detection method thereof.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a recombinant protein based on the extracellular domain of African swine fever virus CD2v protein, comprising: an extracellular domain of the African swine fever virus CD2v protein, wherein an MBP tag protein is inserted at the N terminal of the extracellular domain of the African swine fever virus CD2v protein, or a GCN4 motif or a C-Jun motif is inserted at the C terminal of the extracellular domain of the African swine fever virus CD2v protein.

Further, the extracellular domain of the African swine fever virus CD2v protein is an extracellular domain of a CD2v protein of ASFV 4 type or ASFV 8 type, wherein: the amino acid sequence of the ASFV 4 type CD2v protein extracellular domain is shown in SEQ ID NO: 9, the amino acid sequence of the ASFV 8 type CD2v protein extracellular domain is shown as SEQ ID NO: shown at 10.

Further, the amino acid sequence of the recombinant protein is shown as SEQ ID NO: 1. SEQ ID NO: 2 or SEQ ID NO: 3, wherein: the amino acid sequence of the recombinant protein formed by inserting the MBP tag protein into the N end of the extracellular domain of the CD2v protein is shown as SEQ ID NO: 1, the amino acid sequence of the recombinant protein formed by inserting a GCN4 motif into the C end of the extracellular domain of the CD2v protein is shown as SEQ ID NO: 2, the amino acid sequence of the recombinant protein formed by inserting the C-Jun motif into the C terminal of the extracellular domain of the CD2v protein is shown as SEQ ID NO: 3, respectively.

CD2v is located on the outer membrane of virions and plays an important role in viral transmission, immune regulation and escape. Immunization of animals with homologous CD2v resulted in a protective immune response and prevented infection with homologous ASFV while challenging with chimeric ASFV viruses, suggesting that CD2v may provide serotype-specific protective immunity. In addition, CD2v is very immunogenic and can produce both humoral and cellular immunity. Therefore, the invention finds that the recombinant human immunodeficiency virus (CD2 v) has the potential to be used as a candidate target for serological diagnosis and an ASFV antigen protection target, has the application potential of distinguishing

serotypes

4 and 8 in clinical samples, and glycosylation modification of the CD2v extracellular domain is necessary for ELISA detection of African Swine Fever Virus (ASFV) antibodies.

Based on a general inventive concept, the invention also provides a construction method of a recombinant protein based on the extracellular domain of the African swine fever virus CD2v protein, which comprises the following steps: fusing a nucleotide fragment for coding the extracellular domain of the African swine fever virus CD2v protein with a nucleotide fragment for coding an MBP tag protein, a GCN4 motif or a c-Jun motif, subcloning the fused nucleotide fragment onto a vector, and expressing an obtained expression plasmid in 293F cells, baculovirus or escherichia coli to obtain the recombinant protein.

Further, the sequence of the nucleotide fragment encoding the extracellular domain of the African swine fever virus CD2v protein is shown in SEQ ID NO: 4 or SEQ ID NO: 5, wherein: the sequence of the nucleotide fragment for encoding the extracellular domain of the ASFV 4 type CD2v protein is shown in SEQ ID NO: 4, the sequence of the nucleotide fragment for coding the extracellular domain of the ASFV 8 type CD2v protein is shown as SEQ ID NO: 5, respectively.

Further, the sequence of the nucleotide fragment encoding the MBP tag protein is shown as SEQ ID NO: 6, the sequences of the nucleotide fragments encoding the GCN4 motif are shown as SEQ ID NO: 7, and the sequences of the nucleotide fragments encoding the c-Jun motif are respectively shown in 8.

Based on a general inventive concept, the invention also provides an application of the recombinant protein in establishing an indirect ELISA detection kit or an indirect ELISA detection method.

Further, the process of establishing the indirect ELISA detection kit or the indirect ELISA detection method comprises the following steps: the recombinant protein of any one of claims 1 to 3 or obtained by the construction method of any one of claims 4 to 6 is used as a coating antigen, the recombinant protein is diluted by PBS and then added into a hole of an ELISA plate for coating overnight, then blocking, serum incubation, IgG and IgA HRP enzyme-labeled secondary antibody incubation and color reaction are carried out, the absorbance of a reaction solution at 450nm is measured by an enzyme-labeling instrument, a critical value is calculated according to ELISA data, and the critical value is used as a standard for judging the negative and positive of a subsequent sample and is used for establishing an indirect ELISA detection kit or an indirect ELISA detection method.

Based on a general inventive concept, the present invention also provides an indirect ELISA detection kit, comprising: the kit comprises a 96-well detachable enzyme-linked reaction plate coated with an antigen, positive control serum, negative control serum, a horseradish peroxidase-labeled goat anti-pig secondary antibody (purchased from KPL company, the product number is 14-14-06), a 10-time concentrated PBST cleaning solution, a TMB color development solution (purchased from KPL company, the product number is 5120-0076) and a stop solution, wherein the antigen is the recombinant protein.

Further, the positive control serum is immune serum of the recombinant protein, the negative control serum is pig serum without specific pathogens, the horseradish peroxidase-labeled goat anti-pig secondary antibody is obtained by diluting stock solution of the horseradish peroxidase-labeled goat anti-pig secondary antibody by 1:7000 times of volume, the 10-time concentrated PBST washing solution is phosphate buffer solution containing 0.05% Tween 20 and having a 0.01mol/L, pH value of 7.4, and the stop solution is 2mol/L sulfuric acid solution.

Based on a general inventive concept, the present invention also provides an indirect ELISA detection method, comprising the steps of: the recombinant protein of any one of claims 1 to 3 or obtained by the construction method of any one of claims 4 to 6 is used as a coating antigen, the recombinant protein is diluted by PBS and added into a hole of an ELISA plate for coating overnight, then sealing, incubation of a serum sample to be detected, incubation of an HRP enzyme-labeled antibody of IgG or IgA and color reaction are carried out, the absorbance of a reaction solution at 450nm is measured by an enzyme-labeling instrument, the detection result is judged by referring to the OD450 critical value standard of a detection item, the judgment result that the OD450 is greater than or equal to the critical value is positive, and the judgment result that the OD450 is less than the critical value is negative.

Furthermore, the recombinant protein is obtained by fusing a nucleotide fragment encoding the extracellular domain of the African swine fever virus CD2v protein with a nucleotide fragment encoding a c-Jun motif, subcloning the fused protein onto a vector, and expressing the obtained expression plasmid by 293F cells, namely CD2 v-ED-c-Jun-293.

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

1. the recombinant protein provided by the invention has the advantages that the MBP tag protein is inserted into the N end of the extracellular domain of the African swine fever virus CD2v protein, the GCN4 motif or the C-Jun motif is inserted into the C end of the extracellular domain, the soluble expression and purification can be carried out in expression systems of mammals, baculoviruses and escherichia coli, the recombinant protein has the potential of distinguishing wild viruses from CD2v deletion strains, having serum specificity and distinguishing ASFV 4 types from ASFV 8 types, and the ASFV antibodies (IgG and IgA) can be detected based on ELISA of the CD2v extracellular domain.

2. The recombinant protein is used for ELISA after GCN4 or c-Jun is added into CD2v extracellular domain, compared with the conventional CD2v extracellular domain, the recombinant protein obviously improves the recognition capability of ASFV positive serum samples, and can be used for establishing an indirect ELISA detection kit or an indirect ELISA detection method.

3. The indirect ELISA detection kit and the detection method have higher sensitivity and specificity and wide application.

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 description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is an SDS-PAGE analysis of the extracellular domain of MBP-CD2v purified after preparation of different expression systems from different sources; wherein, M: protein molecular weight Marker; 1: MBP-CD2v-ED-E (about 65 kDa); 2: MBP-CD2v-ED-H5 (about 75 kDa); 3: MBP-CD2v-ED-293 (about 105 kDa).

FIG. 2 is a screen for coating antigens based on different types of recombinant CD2v extracellular domains; CD2v-ED-293 expressed by 293F cells, CD2v-ED-H5 expressed by a baculovirus system and CD2v-ED-E (a) expressed by an escherichia coli expression system, CD2v-ED-c-Jun-293 expressed by 293F cells and CD2v-ED-GCN4-293(b) are used as antigens to respectively detect ASFV positive serum samples; p values were calculated by two-tailed assay, P < 0.05, P < 0.01, P < 0.001.

FIG. 3 shows different types of ASFV antibodies and specificity assays; anti-ASFV, PCV2, PRV, PEDV, PRRSV and CSFV positive sera were tested for (a) IgG and (b) IgA using an ELISA based on CD2v extracellular domain.

FIG. 4 is a graph showing the detection of IgG in standard sera of wild-type strains and CD2v gene-deleted strains at different dilution times by established ELISA; p values were calculated by two-tailed assay, P < 0.0001.

FIG. 5 is the result of serotype specific analysis and cross-reaction with other common porcine pathogens; anti-ASFV (type 8), PCV2, PRV, PEDV, PRRSV and CSFV positive sera were detected using an ELISA method based on the detection of IgG from the extracellular domain of CD2v type 4.

Detailed Description

In order to facilitate an understanding of the invention, reference will now be made in detail to the present preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings, and the scope of the invention is not limited to the following specific embodiments.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

Example (b):

a recombinant protein based on the extracellular domain of African swine fever virus CD2v protein, comprising: the extracellular domain of African swine fever virus CD2v protein, and MBP tag protein inserted into the extracellular domain at the N terminal, GCN4 motif or C-Jun motif at the C terminal. Wherein: the amino acid sequence of the recombinant protein formed by fusing the extracellular domain of the CD2v protein and the MBP tag protein is shown as SEQ ID NO: 1, the amino acid sequence of the recombinant protein formed by fusing the extracellular domain of the CD2v protein and the GCN4 motif is shown as SEQ ID NO: 2, the amino acid sequence of the recombinant protein formed by fusing the extracellular domain of the CD2v protein and the c-Jun motif is shown as SEQ ID NO: 3, respectively.

The construction and screening process of the recombinant protein is as follows:

first, experiment mode

1. Plasmid construction

The sequence of ASFV type 4 BA71 CD2v (GenBank accession No. AKO62740) and ASFV serum type 8 Pig/HLJ/18CD2v (GenBank accession No. QBH90546) were downloaded from NCBI (national Center of Biotechnology information) website, respectively, and the nucleotide fragment was subject to codon optimization for human origin for expression in 293F cells. In addition, other expression systems are respectively subjected to codon optimization, and nucleotide fragments encoding the MBP tag, GCN4 and c-Jun are respectively fused with the ASFV 8 type CD2v extracellular domain so as to be conveniently expressed in mammalian, baculovirus and Escherichia coli expression systems. All nucleotide fragments of ASFV were synthesized by commercialization (Kinsley), and subcloned into vectors.

Wherein, the nucleotide sequence for coding ASFV 4 type CD2v ectodomain is shown in SEQ ID NO: 4, and the nucleotide sequence of the ASFV 8 type CD2v ectodomain is shown as SEQ ID NO: 5, the nucleotide sequences of the nucleotide fragments for encoding the MBP label, GCN4 and c-Jun are respectively shown as SEQ ID NO: 6-8. The amino acid sequence of the ASFV 4 type CD2v extracellular domain is shown in SEQ ID NO: 9, the amino acid sequence of the ASFV 8 type CD2v ectodomain is shown in SEQ ID NO: shown at 10.

2. Protein expression preparation

(1)293 expression System preparation

Several recombinant proteins are prepared in different expression systems respectively and are used for obtaining CD2v ectodomain soluble recombinant proteins prepared by different expression systems, and CD2v ectodomain (CD2v-ED) and CD2v ectodomain for serum type 4 and type 8 recombinant proteinsThe recombinant proteins (serum 8 type) of the ectodomain fused with MBP tags (MBP-CD2v-ED-293), GCN4(CD2v-ED-GCN4-293) and c-Jun (CD2v-ED-c-Jun-293) respectively are subjected to recombinant expression by using a mammalian expression system. The main process is to prepare the expression plasmids of different recombinant proteins by using a plasmid mass extraction kit (OMEGA company), extract to obtain high-purity plasmids without endotoxin, and measure the concentration. 293F cells transfected with endotoxin-free plasmid DNA at 5% CO ₂ The culture was carried out at 37 ℃ and 140rpm, and the supernatant was collected after culturing in SMM 293T-II expression medium (Yinqianzhou Co.) for 5-7 days.

(2) Preparation of insect baculovirus expression System

The recombinant protein (serotype 8) with the extracellular domain of CD2v fused to the MBP tag (MBP-CD2v-ED-H5) was also expressed in a baculovirus expression system and then expressed using a pFastBac baculovirus expression system (Invitrogen). Briefly, recombinant bacmid DNA was generated after transposing the constructed recombinant plasmid into DH10Bac competent cells. Recombinant bacmid DNA was transfected into Sf9 cells, recombinant baculovirus was generated and amplified. Then, the recombinant baculovirus was infected with High Five cells cultured in SIM HF (seupizhou corporation) medium, cultured at 27 ℃ at 130rpm, and the supernatant was collected after 72 hours.

(3) Preparation of E.coli expression System

The main steps of expressing a recombinant protein (serotype 8) with a CD2v ectodomain fused MBP tag (MBP-CD2v-ED-E) in an Escherichia coli expression system are that a recombinant plasmid is converted into BL21(DE3) competent cells, after a large amount of induction expression is carried out by enlarging the culture volume, the supernatant is removed after 5000g centrifugation for 10 minutes, a bacterial pellet is harvested, and then the cell pellet is subjected to a culture medium containing 50mM NaH ₂ PO ₄ ·2H ₂ O, 20mM imidazole, 10mM Tris base, 300mM NaCl and 0.1% Triton X-100. After breaking the cells by ultrasonic on ice, centrifuging at 4 ℃ and 20000g for 20min, and harvesting the supernatant to realize soluble expression. The 5 recombinant proteins are respectively derived from a mammal expression system, a baculovirus expression system and an escherichia coli expression system, can be purified from supernatant by adopting a nickel column (GE Healthcare), and the protein after protein purification is dissolved after being concentratedThe solution was stored in 10mM PBS, pH 7.4, until use.

3. Screening for antigens coating ELISA

In order to confirm which system prepared recombinant protein from CD2v recombinant protein expressed by a mammalian expression system, a baculovirus expression system and an Escherichia coli expression system is more suitable for establishing an ELISA scheme, wherein the serum incubation time, the HRP enzyme-labeled secondary antibody incubation time and the TMB reaction time are set and determined according to a conventional method, and the specific operation steps of other experimental methods are as follows:

(1) coating: the recombinant proteins obtained from 3 different expression systems were diluted with PBS (10mM, pH 7.4), applied to a 96-well ELISA plate (Corning Corp.), and coated overnight at 4 ℃.

(2) And (3) sealing: after completion of the coating, the well was drained, washed 5 times with 300 μ L PBST, blocked with BSA, incubated at room temperature for 3h, washed 5 times with PBST, air dried and sealed, and stored at 2-8 ℃ in the dark.

(3) Incubation serum: mu.L of diluted pig serum was added to each well and incubated for 1h at room temperature.

(4) Incubation of HRP enzyme-labeled secondary antibody: after completion of the serum incubation, washed 5 times with PBST, 100 μ L of diluted HRP enzyme-labeled goat anti-porcine IgG (h + L) (KPL corporation) was added to the wells and incubated at room temperature for 30 minutes.

(5) And (3) color development reaction: after completion of incubation with HRP enzyme-labeled secondary antibody, the cells were washed 5 times with PBST, 50. mu.L of TMB coloring solution (KPL Co.) was added to each well, and after incubation at room temperature for 5 minutes, 2M H was added to each well ₂ SO ₄ The absorbance of each reaction well at 450nm was measured by a microplate reader.

4. Determination of methods for detecting IgG and IgA

The recombinant protein at a concentration determined to be coated was diluted with PBS (10mM, pH 7.4), and then added to each of 96-well ELISA plates (Corning Corp.) and coated overnight at 4 ℃. Subsequent experimental work steps were then performed:

(1) and (3) sealing: after completion of the coating, the well was drained, washed 5 times with 300 μ L PBST, blocked with BSA, incubated at room temperature for 3h, washed 5 times with PBST, air dried and sealed, and stored at 2-8 ℃ in the dark.

(2) Incubation serum: mu.L of diluted pig serum was added to each well and incubated for 1h at room temperature.

(3) Incubation of HRP enzyme-labeled secondary antibody: after completion of the serum incubation, washed 5 times with PBST, 100 μ L of diluted HRP enzyme-labeled goat anti-porcine IgG (H + L) (KPL company) and HRP enzyme-labeled goat anti-porcine IgA (H + L) (Abcam, uk) were added to different wells and incubated at room temperature for 30 minutes for subsequent detection of IgG and IgA.

(4) And (3) color development reaction: after completion of incubation with HRP enzyme-labeled secondary antibody, the cells were washed 5 times with PBST, 50. mu.L of TMB coloring solution (KPL Co.) was added to each well, and after incubation at room temperature for 5 minutes, 2M H was added to each well ₂ SO ₄ And (3) measuring the absorbance of each reaction hole at 450nm by using an enzyme-linked immunosorbent assay (ELISA) instrument, and calculating a critical value according to ELISA data to be used as a standard for judging the negative and positive of a subsequent sample.

5. Specificity test

Selecting 50 ASFV clinical negative control sera (collected before domestic outbreak of African swine fever in 2018), detecting IgG and IgA by using the established ELISA method, and judging the specificity of the ELISA method according to the obtained absorbance value result and a critical value.

6. Cross reaction test

Clinical serum samples which are determined to be positive to antibodies of porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), Porcine Epidemic Diarrhea (PEDV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Classical Swine Fever Virus (CSFV) are detected by an established ELISA method, whether positive serum of different pathogens can react with recombinant protein coated on an ELISA plate or not is judged according to an obtained absorbance value result and a critical value, and the scheme is respectively determined to be used for detecting the cross reaction condition of IgG and IgA of antibodies of ASFV CD2 v.

7. Compliance rate test

51 parts of ASFV positive clinical serum and 45 parts of clinical negative serum provided by the animal epidemic disease prevention control center in Hunan province are selected, an ELISA method established by the inventor is used for carrying out IgG and IgA test, the result is judged according to the obtained light absorption value result and the critical value, and the coincidence rate of IgG and IgA detection in the aspect of clinical serum sample identification is calculated.

8. Antibody for distinguishing wild virus and CD2v deletion strain based on ELISA

2 standard serum samples purchased from ASFV 8 type wild strains and CD2v gene deletion strains immunized by Chinese veterinary drug inspection are selected as standard positive control, IgG is detected by using the kit, the result is judged according to the obtained light absorption value result and the critical value, and the condition of distinguishing different strains is determined.

9. Resolution of ASFV serotypes based on ELISA

The purified recombinant protein of CD2v serotype 4 was used as a coating antigen, diluted with PBS (10mM, pH 7.4), and then applied to a 96-well ELISA plate (Corning Co.) and coated overnight at 4 ℃. Subsequent experimental work steps were then carried out:

(1) and (3) sealing: after coating was complete, the wells were drained, washed 5 times with 300 μ L PBST and blocked with BSA, and incubated at room temperature for 3 h. Washed 5 times with PBST, air dried and sealed, and stored at 2-8 ℃ in the dark.

(2) Incubation serum: mu.L of diluted swine serum including African swine fever serotype 8 (ASFV) porcine pseudorabies virus (PRV), Porcine Epidemic Diarrhea (PEDV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Classical Swine Fever Virus (CSFV) antibody positive samples were added to each well and incubated for 1h at room temperature, respectively.

(3) Incubation of HRP enzyme-labeled secondary antibody: after completion of the serum incubation, washed 5 times with PBST, 100 μ L of diluted HRP enzyme-labeled goat anti-porcine IgG (h + L) (KPL company) was added to the wells and incubated at room temperature for 30 minutes.

(4) And (3) color development reaction: after the incubation with HRP enzyme-labeled secondary antibody was completed, the reaction mixture was washed 5 times with PBST, 50. mu.L of a TMB color developing solution (KPL Co.) was added to each well, and after incubation at room temperature for 5 minutes, 2M H was added to each well ₂ SO ₄ And (3) measuring the absorbance of each reaction hole at 450nm by using an enzyme-linked immunosorbent assay (ELISA) instrument, calculating a critical value according to ELISA data, and using the critical value as a standard for judging the negative and positive of a subsequent sample, namely: the determination result is positive when OD450 is greater than or equal to the critical value, and negative when OD450 is less than the critical value.

Second, experimental results

1. Recombinant protein production for different expression systems

Through the purification of the MBP-CD2v extracellular domain recombinant proteins from different expression systems by using a nickel column, in the analysis of SDS-PAGE, the recombinant protein MBP-CD2v-ED-E from an Escherichia coli expression system is about 65kDa (Line 1), the recombinant protein MBP-CD2v-ED-H5 from an insect baculovirus expression system is about 75kDa (Line 2), and the recombinant protein MBP-CD2v-ED-293 from a 293 expression system is about 105kDa (Line 3), which are respectively expressed in Escherichia coli, High Five cells and 293F cells. As a result of SDS-PAGE analysis, it was found that the molecular weights of the recombinant proteins MBP-CD2v-ED-293, MBP-CD2v-ED-H5 and MBP-CD2v-ED-E differ due to differences in glycosylation modification in different expression systems (FIG. 1).

2. Screening of recombinant proteins prepared from different expression systems for ELISA coating

The multiple recombinant proteins are respectively used as coating antigens for ELISA plate coating. As expected, ASFV positive serum samples recognized CD2v recombinant protein from all three expression systems. However, the optical density of the 450nm (OD450) values (p < 0.01) for the recombinant protein MBP-CD2v-ED-293 prepared on the basis of the 293 expression system and the recombinant protein MBP-CD2v-ED-H5 prepared on the basis of the insect baculovirus expression system were higher than that for the recombinant protein MBP-CD2v-ED-E prepared on the basis of the E.coli expression system, while there was no significant difference in the detection of positive samples between MBP-CD2v-ED-293 and MBP-CD2v-ED-H5 (FIG. 2). Furthermore, the MBP tag used in the ELISA had a high background value close to 0.5, much higher than the blank control (p < 0.01), resulting in recombinant MBP-CD2v-ED (serotype 8) not being used in the ELISA due to the use of the MBP tag (fig. 2 a). Therefore, we used the MBP tag-free recombinant protein CD2v-ED-293 in subsequent assays. Comparing CD2v-ED-293 prepared by 293 cells with CD2v-ED-c-Jun-293 and CD2v-ED-GCN4-293, the ELISA coated by C D2v-ED-293 is found to have the lowest OD450 value when detecting ASFV positive samples and has extremely obvious difference (p is less than 0.01) in the light absorption values corresponding to the other two recombinant proteins, and then an ELI SA scheme is established on the basis of the CD2v-ED-c-Jun-293 recombinant protein.

Based on the above, the indirect ELISA detection kit defined by the invention comprises the following components: the kit comprises a 96-well detachable enzyme-linked reaction plate coated with antigen (the antigen is CD2v-ED-c-Jun-293 recombinant protein), positive control serum (recombinant protein immune serum), negative control serum (swine serum without specific pathogen), horseradish peroxidase-labeled goat anti-swine secondary antibody (purchased from KPL company, the product number is 14-14-06, the stock solution is diluted 1:7000 times), 10-fold concentrated PBST cleaning solution (phosphate buffer solution containing 0.01mol/L, pH value of 0.05% Tween 20 and having 7.4), TMB color development solution (purchased from KPL company, the product number is 5120-. In fact, the coating antigen can also be other recombinant proteins such as CD2v-ED-293, CD2v-ED-GCN4-293 and the like.

3. Determination of critical value of IgG and IgA detection and calculation of specificity

Our ELISA detection method can detect IgG and IgA, respectively, by changing the secondary antibody. Detection of 50 negative control sera (collected prior to 2018 domestic outbreak of African swine fever) was further confirmed with an anti-ASFV-specific IgG commercial kit. All negative control sera were tested for IgG and IgA using two enzyme-linked immunosorbent assays (ELISA) and the cut-off values were determined from the negative sera. Calculating the mean value of OD450 values

And Standard Deviation (SD), and the critical value is defined as

The OD450 cutoff values for IgG and IgA, calculated from the OD values of negative serum samples, were 0.39 and 0.34, respectively, for the subsequent sample determinations, i.e.: in the IgG negative and positive judgment, the IgG is judged to be positive if the OD450 is more than or equal to 0.39, and the IgG is judged to be negative if the OD450 is less than 0.39; in the positive and negative IgA determination, the determination result was positive at OD 450. gtoreq.0.34, and negative at OD450 < 0.34. IgG and IgA specificities were 100% (50/50) and 88.0% (44/50), respectively. Overall, the most specific method is to detect IgG in serum samples.

4. Cross reactivity test

Specific detection two ELISA methods are adopted to detect positive serum of anti-PCV 2, PRV, PEDV, PRRSV and CSFV respectively. The serum samples detected by both ELISA did not reach the set critical value, indicating no cross-reaction with other common porcine pathogens (FIG. 3).

5. Compliance testing

A commercial kit is selected to test 51 clinical sera which are ASFV positive and 45 clinical negative sera, IgG and IgA are tested by an ELISA method established by people to test 51 clinical sera which are infected with ASFV positive, 45 clinical negative sera and 50 negative control sera collected before 2018 domestic African swine fever outbreak, all 51 clinical sera samples infected with ASFV are tested by ELISA to be IgG positive and 45 clinical negative sera, 5 parts of ELISA kit are judged to be positive, 50 parts of negative control serum collected before 2018 domestic African swine fever outbreak are all negative, therefore, the coincidence rate is 96.7% [ (51+95)/(51+0+5+95) ], and in the same way, however, since the IgA does not have a commercial kit, only the detection result of IgG can be used for reference, and the coincidence rate of IgG positive samples for IgA detection is 78.1% [ (51+95)/(51+ 27+14+95) ].

6. Antibody for distinguishing wild virus and CD2v deletion strain

2 standard serum samples immunized with ASFV 8 type wild strain and CD2v gene deletion strain were selected. When different times of diluted standard serum are detected, the serum concentration of the wild strain is still positive when reaching 1:10240, and the serum concentration of the strain lacking the CD2v gene is always lower than the critical value and is judged to be negative (figure 4). Our established ELISA assay based on the extracellular domain of CD2v can be used to distinguish antibodies from wild-type and CD2v gene-deleted strains.

7. Differentiating ASFV serotypes without cross-reacting with other pathogens

After coating with extracellular domain of type 4CD2v on ELISA plates, which were subsequently used to detect ASFV positive sera (type 8 serum) and anti-PCV 2, PRV, PEDV, PRRSV and CSFV positive serum samples, the absorbance values obtained were all below the cut-off value and no positive detection was obtained (fig. 5). Clearly, the CD2v extracellular domain has serotype specific differences in the type 4 ASFV and type 8 ASFV IgG assays. In addition, the extracellular domain of serotype 4CD2v did not cross-react with other common porcine pathogens.

In conclusion, the recombinant protein of the invention, namely the insertion of the MBP tag protein at the N-terminal and the insertion of the GCN4 motif or C-Jun motif at the C-terminal of the extracellular domain of the African swine fever virus CD2v protein can perform soluble expression and purification in expression systems of mammals, baculoviruses and Escherichia coli, has the potential of distinguishing wild viruses from CD2v deletion strains, has serum specificity and distinguishing ASFV type 4 and type 8 serological types, and can detect ASFV antibodies (IgG and IgA) based on ELISA of the CD2v extracellular domain (CD2 v-ED-293). The kit is used for ELISA after GCN4 or c-Jun is added into CD2v extracellular domain, compared with the conventional CD2v extracellular domain, the recognition capability of the kit on ASFV positive serum sample is obviously improved, and the kit can be used for establishing an indirect ELISA detection kit or an indirect ELISA detection method. The indirect ELISA detection kit and the detection method established by the kit have higher sensitivity and specificity and are widely applied.

Sequence listing

<110> Hunan Intelligent Biotechnology Ltd

<120> recombinant protein based on extracellular domain of African swine fever virus CD2v protein, and construction method and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 578

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Gly Ser His His His His His His Gly Ser Leu Gln Asp Lys Ile

1 5 10 15

Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn

20 25 30

Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys

35 40 45

Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val

50 55 60

Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg

65 70 75 80

Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp

85 90 95

Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg

100 105 110

Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser

115 120 125

Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu

130 135 140

Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala

145 150 155 160

Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala

165 170 175

Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile

180 185 190

Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe

195 200 205

Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr

210 215 220

Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile

225 230 235 240

Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr

245 250 255

Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe

260 265 270

Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu

275 280 285

Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu

290 295 300

Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser

305 310 315 320

Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu

325 330 335

Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala

340 345 350

Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg

355 360 365

Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asp Tyr Asp Ile

370 375 380

Pro Thr Thr Ile Asp Tyr Trp Val Ser Phe Asn Lys Thr Ile Ile Leu

385 390 395 400

Asp Ser Asn Ile Thr Asn Asp Asn Asn Asp Ile Asn Gly Val Ser Trp

405 410 415

Asn Phe Phe Asn Asn Ser Phe Asn Thr Leu Ala Thr Cys Gly Lys Ala

420 425 430

Gly Asn Phe Cys Glu Cys Ser Asn Tyr Ser Thr Ser Ile Tyr Asn Ile

435 440 445

Thr Asn Asn Cys Ser Leu Thr Ile Phe Pro His Asn Asp Val Phe Asp

450 455 460

Thr Thr Tyr Gln Val Val Trp Asn Gln Ile Ile Asn Tyr Thr Ile Lys

465 470 475 480

Leu Leu Thr Pro Ala Thr Pro Pro Asn Ile Thr Tyr Asn Cys Thr Asn

485 490 495

Phe Leu Ile Thr Cys Lys Lys Asn Asn Gly Thr Asn Thr Asn Ile Tyr

500 505 510

Leu Asn Ile Asn Asp Thr Phe Val Lys Tyr Thr Asn Glu Ser Ile Leu

515 520 525

Glu Tyr Asn Trp Asn Asn Ser Asn Ile Asn Asn Phe Thr Ala Thr Cys

530 535 540

Ile Ile Asn Asn Thr Ile Ser Thr Ser Asn Glu Thr Thr Leu Ile Asn

545 550 555 560

Cys Thr Tyr Leu Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe Phe Lys

565 570 575

Leu Tyr

<210> 2

<211> 238

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gly Ser His His His His His His Gly Ser Ile Asp Tyr Trp Val

1 5 10 15

Ser Phe Asn Lys Thr Ile Ile Leu Asp Ser Asn Ile Thr Asn Asp Asn

20 25 30

Asn Asp Ile Asn Gly Val Ser Trp Asn Phe Phe Asn Asn Ser Phe Asn

35 40 45

Thr Leu Ala Thr Cys Gly Lys Ala Gly Asn Phe Cys Glu Cys Ser Asn

50 55 60

Tyr Ser Thr Ser Ile Tyr Asn Ile Thr Asn Asn Cys Ser Leu Thr Ile

65 70 75 80

Phe Pro His Asn Asp Val Phe Asp Thr Thr Tyr Gln Val Val Trp Asn

85 90 95

Gln Ile Ile Asn Tyr Thr Ile Lys Leu Leu Thr Pro Ala Thr Pro Pro

100 105 110

Asn Ile Thr Tyr Asn Cys Thr Asn Phe Leu Ile Thr Cys Lys Lys Asn

115 120 125

Asn Gly Thr Asn Thr Asn Ile Tyr Leu Asn Ile Asn Asp Thr Phe Val

130 135 140

Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn Trp Asn Asn Ser Asn

145 150 155 160

Ile Asn Asn Phe Thr Ala Thr Cys Ile Ile Asn Asn Thr Ile Ser Thr

165 170 175

Ser Asn Glu Thr Thr Leu Ile Asn Cys Thr Tyr Leu Thr Leu Ser Ser

180 185 190

Asn Tyr Phe Tyr Thr Phe Phe Lys Leu Tyr Gly Gly Ser Arg Met Lys

195 200 205

Gln Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn Tyr His Leu

210 215 220

Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly Glu Arg

225 230 235

<210> 3

<211> 241

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Gly Ser His His His His His His Gly Ser Ile Asp Tyr Trp Val

1 5 10 15

Ser Phe Asn Lys Thr Ile Ile Leu Asp Ser Asn Ile Thr Asn Asp Asn

20 25 30

Asn Asp Ile Asn Gly Val Ser Trp Asn Phe Phe Asn Asn Ser Phe Asn

35 40 45

Thr Leu Ala Thr Cys Gly Lys Ala Gly Asn Phe Cys Glu Cys Ser Asn

50 55 60

Tyr Ser Thr Ser Ile Tyr Asn Ile Thr Asn Asn Cys Ser Leu Thr Ile

65 70 75 80

Phe Pro His Asn Asp Val Phe Asp Thr Thr Tyr Gln Val Val Trp Asn

85 90 95

Gln Ile Ile Asn Tyr Thr Ile Lys Leu Leu Thr Pro Ala Thr Pro Pro

100 105 110

Asn Ile Thr Tyr Asn Cys Thr Asn Phe Leu Ile Thr Cys Lys Lys Asn

115 120 125

Asn Gly Thr Asn Thr Asn Ile Tyr Leu Asn Ile Asn Asp Thr Phe Val

130 135 140

Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn Trp Asn Asn Ser Asn

145 150 155 160

Ile Asn Asn Phe Thr Ala Thr Cys Ile Ile Asn Asn Thr Ile Ser Thr

165 170 175

Ser Asn Glu Thr Thr Leu Ile Asn Cys Thr Tyr Leu Thr Leu Ser Ser

180 185 190

Asn Tyr Phe Tyr Thr Phe Phe Lys Leu Tyr Gly Gly Ser Leu Glu Glu

195 200 205

Lys Val Lys Thr Leu Lys Ala Gln Asn Ser Glu Leu Ala Ser Thr Ala

210 215 220

Asn Met Leu Arg Glu Gln Val Ala Gln Leu Lys Gln Lys Val Met Asn

225 230 235 240

His

<210> 4

<211> 603

<212> DNA

<213> African swine fever virus (African swine fever virus)

<400> 4

atgggaagcc accaccacca ccaccacggg tccaacatta tcatctggtc caccctgaac 60

cagaccgtgt tcctgaacaa catctttaca attaatgaca catacggcgg actgttctgg 120

aacacatact acgacaacaa ccggagcaac ttcacctatt gtggcatcgc cggcaattac 180

tgcagctgct gcggccacaa tatcagcctt tataatacga ccaacaactg cagcctgatc 240

atctttccaa acaatacaga aatcttcaac agaacctacg agctggtgta cctggacaag 300

aaaatcaact acaccgttaa gctgctcaaa agcgtggaca gccctaccat cacctacaac 360

tgtacaaaca gcctgattac atgtaaaaac aacaacggca caaacgtgaa catctacctg 420

atcatcaaca ataccatcgt gaacgacacc aatggagata tcctgaacta ctactggaac 480

ggcaacaaca atttcaccgc cacatgcatg atcaacaaca ccatcagcag cctgaatgag 540

acagagaaca tcaactgcac caaccccatc ctgaagtacc agaactacct gagcaccctg 600

taa 603

<210> 5

<211> 609

<212> DNA

<213> African swine fever virus (African swine fever virus)

<400> 5

atgggaagcc accaccacca ccaccacggg tccatcgact actgggtgtc tttcaataag 60

acaatcatcc tggatagcaa catcaccaat gacaacaatg atatcaacgg cgtgagctgg 120

aatttcttta acaattcctt caacaccctg gccacatgcg gcaaggccgg caacttttgc 180

gagtgttcta attactctac cagcatctat aacatcacaa acaattgtag cctgaccatc 240

ttcccacaca atgacgtgtt tgataccaca taccaggtgg tgtggaacca gatcatcaat 300

tatacaatca agctgctgac ccctgccaca ccccctaaca tcacctacaa ctgcacaaat 360

tttctgatca cctgtaagaa gaacaatggc accaacacaa atatctatct gaacatcaat 420

gacaccttcg tgaagtacac aaatgagtcc atcctggagt acaactggaa caactctaac 480

atcaacaact tcaccgccac atgcatcatc aacaatacca tctccacatc taacgagacc 540

acactgatca attgtaccta cctgacactg agctccaact acttctatac cttctttaag 600

ctgtactaa 609

<210> 6

<211> 1161

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgggaagcc accaccacca ccaccacggg tccctgcagg acaaaatcga agaaggtaaa 60

ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa 120

ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa 180

ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc 240

tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag 300

gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac 360

ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca 420

aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg 480

ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt 540

tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct 600

ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca 660

gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc 720

aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta 780

ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt 840

aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact 900

gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct 960

tacgaggaag agttggcgaa agatccacgt attgccgcca ccatggaaaa cgcccagaaa 1020

ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg 1080

gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact 1140

gattacgata tcccaacgac c 1161

<210> 7

<211> 99

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cggatgaagc aactggaaga taaggttgag gaactgctga gcaagaacta ccacctggag 60

aacgaggttg ccagactgaa aaaactcgtg ggcgagcgg 99

<210> 8

<211> 108

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctggaagaga aggtcaagac cctgaaggct cagaactccg agctggccag cacagccaat 60

atgctgcggg aacaggtggc ccagctgaag cagaaggtga tgaaccac 108

<210> 9

<211> 190

<212> PRT

<213> African swine fever virus (African swine fever virus)

<400> 9

Met Asn Ile Ile Ile Trp Ser Thr Leu Asn Gln Thr Val Phe Leu Asn

1 5 10 15

Asn Ile Phe Thr Ile Asn Asp Thr Tyr Gly Gly Leu Phe Trp Asn Thr

20 25 30

Tyr Tyr Asp Asn Asn Arg Ser Asn Phe Thr Tyr Cys Gly Ile Ala Gly

35 40 45

Asn Tyr Cys Ser Cys Cys Gly His Asn Ile Ser Leu Tyr Asn Thr Thr

50 55 60

Asn Asn Cys Ser Leu Ile Ile Phe Pro Asn Asn Thr Glu Ile Phe Asn

65 70 75 80

Arg Thr Tyr Glu Leu Val Tyr Leu Asp Lys Lys Ile Asn Tyr Thr Val

85 90 95

Lys Leu Leu Lys Ser Val Asp Ser Pro Thr Ile Thr Tyr Asn Cys Thr

100 105 110

Asn Ser Leu Ile Thr Cys Lys Asn Asn Asn Gly Thr Asn Val Asn Ile

115 120 125

Tyr Leu Ile Ile Asn Asn Thr Ile Val Asn Asp Thr Asn Gly Asp Ile

130 135 140

Leu Asn Tyr Tyr Trp Asn Gly Asn Asn Asn Phe Thr Ala Thr Cys Met

145 150 155 160

Ile Asn Asn Thr Ile Ser Ser Leu Asn Glu Thr Glu Asn Ile Asn Cys

165 170 175

Thr Asn Pro Ile Leu Lys Tyr Gln Asn Tyr Leu Ser Thr Leu

180 185 190

<210> 10

<211> 192

<212> PRT

<213> African swine fever virus (African swine fever virus)

<400> 10

Met Ile Asp Tyr Trp Val Ser Phe Asn Lys Thr Ile Ile Leu Asp Ser

1 5 10 15

Asn Ile Thr Asn Asp Asn Asn Asp Ile Asn Gly Val Ser Trp Asn Phe

20 25 30

Phe Asn Asn Ser Phe Asn Thr Leu Ala Thr Cys Gly Lys Ala Gly Asn

35 40 45

Phe Cys Glu Cys Ser Asn Tyr Ser Thr Ser Ile Tyr Asn Ile Thr Asn

50 55 60

Asn Cys Ser Leu Thr Ile Phe Pro His Asn Asp Val Phe Asp Thr Thr

65 70 75 80

Tyr Gln Val Val Trp Asn Gln Ile Ile Asn Tyr Thr Ile Lys Leu Leu

85 90 95

Thr Pro Ala Thr Pro Pro Asn Ile Thr Tyr Asn Cys Thr Asn Phe Leu

100 105 110

Ile Thr Cys Lys Lys Asn Asn Gly Thr Asn Thr Asn Ile Tyr Leu Asn

115 120 125

Ile Asn Asp Thr Phe Val Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr

130 135 140

Asn Trp Asn Asn Ser Asn Ile Asn Asn Phe Thr Ala Thr Cys Ile Ile

145 150 155 160

Asn Asn Thr Ile Ser Thr Ser Asn Glu Thr Thr Leu Ile Asn Cys Thr

165 170 175

Tyr Leu Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe Phe Lys Leu Tyr

180 185 190

Claims

1. A recombinant protein based on the extracellular domain of African swine fever virus CD2v protein, which comprises: an extracellular domain of the African swine fever virus CD2v protein, wherein an MBP tag protein is inserted at the N terminal of the extracellular domain of the African swine fever virus CD2v protein, or a GCN4 motif or a C-Jun motif is inserted at the C terminal of the extracellular domain of the African swine fever virus CD2v protein.

2. The recombinant protein based on the extracellular domain of African swine fever virus CD2v protein, according to claim 1, wherein the extracellular domain of African swine fever virus CD2v protein is the extracellular domain of CD2v protein of ASFV type 4 or ASFV type 8.

3. The recombinant protein based on the extracellular domain of African swine fever virus CD2v protein of claim 1 or 2, wherein the amino acid sequence of the recombinant protein formed by inserting MBP tag protein into the N terminal of the extracellular domain of CD2v protein is shown as SEQ ID NO: 1, the amino acid sequence of the recombinant protein formed by inserting a GCN4 motif into the C end of the extracellular domain of the CD2v protein is shown as SEQ ID NO: 2, the amino acid sequence of the recombinant protein formed by inserting the C-Jun motif into the C terminal of the extracellular domain of the CD2v protein is shown as SEQ ID NO: 3, respectively.

4. The method for constructing the recombinant protein based on the extracellular domain of the African swine fever virus CD2v protein, which is described in any one of claims 1-3, and comprises the following steps: fusing a nucleotide fragment for coding the extracellular domain of the African swine fever virus CD2v protein with a nucleotide fragment for coding an MBP tag protein, a GCN4 motif or a c-Jun motif, subcloning the fused nucleotide fragment onto a vector, and expressing an obtained expression plasmid in 293F cells, baculovirus or escherichia coli to obtain the recombinant protein.

5. The construction method according to claim 4, wherein the sequence of the nucleotide fragment encoding the extracellular domain of African swine fever virus CD2v protein is shown as SEQ ID NO: 4 or SEQ ID NO: 5, respectively.

6. The method according to claim 4 or 5, wherein the sequence of the nucleotide fragment encoding the MBP tag protein is as shown in SEQ ID NO: 6, the sequences of the nucleotide fragments encoding the GCN4 motif are shown as SEQ ID NO: 7, and the sequences of the nucleotide fragments encoding the c-Jun motif are shown as SEQ ID NO: shown in fig. 8.

7. Use of a recombinant protein according to any one of claims 1 to 3 or obtained by a method of construction according to any one of claims 4 to 6 for the establishment of an indirect ELISA detection method.

8. The use according to claim 7, wherein the process of establishing an indirect ELISA detection method comprises the steps of: the recombinant protein of any one of claims 1 to 3 or obtained by the construction method of any one of claims 4 to 6 is used as a coating antigen, the recombinant protein is diluted by PBS and then added into a hole of an ELISA plate for coating overnight, then blocking, serum incubation, IgG and IgA HRP enzyme-labeled secondary antibody incubation and color reaction are carried out, the absorbance of a reaction solution at 450nm is measured by an enzyme-labeled instrument, a critical value is calculated according to ELISA data, and the critical value is used as a standard for judging the negative and positive of a subsequent sample to establish an indirect ELISA detection method.

9. An indirect ELISA detection kit, comprising: a 96-well detachable enzyme-linked reaction plate coated with an antigen, positive control serum, negative control serum, a goat anti-pig secondary antibody marked by horseradish peroxidase, a PBST cleaning solution, a TMB color development solution and a stop solution, wherein the antigen is the recombinant protein described in any one of claims 1 to 3 or obtained by the construction method described in any one of claims 4 to 6.

10. An indirect ELISA detection method, characterized by comprising the following steps: the recombinant protein of any one of claims 1 to 3 or obtained by the construction method of any one of claims 4 to 6 is used as a coating antigen, the recombinant protein is diluted by PBS and added into a hole of an ELISA plate for coating overnight, then sealing, incubation of a serum sample to be detected, incubation of an HRP enzyme-labeled antibody of IgG or IgA and color reaction are carried out, the absorbance of a reaction solution at 450nm is measured by an enzyme-labeling instrument, the detection result is judged by referring to the OD450 critical value standard of a detection item, the judgment result that the OD450 is greater than or equal to the critical value is positive, and the judgment result that the OD450 is less than the critical value is negative.