CN113278066A

CN113278066A - Full-swine-origin African swine fever virus monoclonal antibody and preparation method thereof

Info

Publication number: CN113278066A
Application number: CN202110573254.2A
Authority: CN
Inventors: 郑海学; 曹丽艳; �田宏; 王�琦; 孔祥雨; 宋锐; 石正旺
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2021-08-20
Anticipated expiration: 2041-05-25
Also published as: CN113278066B

Abstract

The invention belongs to the technical field of biology, and particularly relates to a full-swine-origin African swine fever virus monoclonal antibody, and a preparation method and application thereof. The monoclonal antibody comprises a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO 1 or SEQ ID NO 5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO 2 or SEQ ID NO 6. The swine-origin monoclonal antibody specific to African swine fever virus retains natural pairing of light chain and heavy chain variable regions, has the advantages of good gene diversity, swine-origin, good antibody affinity, strong specificity and the like, and can be efficiently expressed in a eukaryotic expression system. The invention provides a novel raw material for developing a diagnosis kit of African swine fever virus and also provides a new technical support for developing preventive and therapeutic drugs of the full-swine-derived monoclonal antibody.

Description

Full-swine-origin African swine fever virus monoclonal antibody and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a full-swine-origin African swine fever virus monoclonal antibody and a method for preparing the monoclonal antibody by utilizing a high-throughput single B cell PCR technology.

Background

African Swine Fever (ASF) is an acute and highly contagious infectious disease of pigs caused by African Swine Fever Virus (ASFV), and has short course and 100% mortality. The world health Organization (OIE) ranks the animal epidemic disease as a legal report, and China ranks the animal epidemic disease as a type of animal epidemic disease. The ASF is discovered in Kenya in 1921 for the first time, is introduced into China in 2018 in 8 months, and then rapidly spreads to a plurality of provinces and cities in China, so that huge economic losses are caused to the development of the pig industry in China. Until now, there is no effective commercial vaccine prevention and control ASF, and early diagnosis and regional prevention and control management are relied on in a short time for prevention and control.

ASFV has a large genome structure and a complex immune escape mechanism, and the functions of many proteins are unknown at present. In order to deeply research the role of ASFV coding protein in virus infection, more antibodies capable of identifying the protein need to be developed, and the interaction between the protein and the antibody needs to be deeply researched, so that a key epitope is excavated, accurate diagnosis of ASFV infection is realized, vaccine design is optimized, and a new direction and a new thought are provided for ASF prevention and treatment.

The invention patents with publication numbers CN112592401A and CN112500478A disclose ScFv antibodies VH-VL λ 11 and VH-VL λ 6 against african swine fever virus, respectively, and a preparation method thereof, wherein the antibodies VH-VL λ 11 and VH-VL λ 6 are both antibodies against african swine fever virus, but the antibodies are single chain antibodies, and the single chain antibodies only include heavy chain variable regions and light chain variable regions, and do not include constant regions.

The high-throughput single B cell PCR antibody technology is a novel antibody preparation technology developed in recent years. The technology amplifies antibody genes from a single B cell and performs in-vitro cloning expression, and the antibody obtained by the method keeps the natural pairing of the light chain variable region and the heavy chain variable region and has the advantages of high efficiency, full-natural source, richer gene diversity and the like.

Disclosure of Invention

The invention provides a full-swine-origin African swine fever virus monoclonal antibody and a method for preparing the monoclonal antibody by utilizing a high-throughput single B cell PCR technology.

The first aspect of the invention provides a monoclonal antibody of African swine fever virus of whole pig origin, which comprises a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1 or SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2 or SEQ ID NO. 6. The amino acid sequences of the heavy chain constant region, Kappa light chain constant region and Lambda light chain constant region are shown as SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, respectively.

In a second aspect, the invention provides a DNA fragment encoding the whole swine African swine fever virus monoclonal antibody.

The DNA segment of the heavy chain variable region of the monoclonal antibody of the full-porcine African swine fever virus is shown as SEQ ID NO. 3 or SEQ ID NO. 7, and the DNA segment of the light chain variable region of the monoclonal antibody of the full-porcine African swine fever virus is shown as SEQ ID NO. 4 or SEQ ID NO. 8.

In a third aspect, the invention provides a recombinant expression vector comprising the DNA fragment.

In a fourth aspect, the invention provides a host cell comprising said recombinant expression vector.

The preparation method of the full-swine-origin African swine fever virus monoclonal antibody comprises the following steps:

(1) PBMC is obtained by separating peripheral blood of a naturally infected African swine fever immune-tolerant pig, B lymphocyte sorting is carried out by utilizing flow cytometry, and single ASFV antigen specificity B lymphocyte is separated into a hole of a 96-hole plate;

(2) taking the RNA of the PBMC or the single B lymphocyte separated in the step (1) as a template, and performing reverse transcription on the RNA by utilizing Oligo-dt or a random primer to synthesize cDNA;

(3) amplifying coding genes of heavy chain and light chain constant regions of the antibody by using the cDNA of the PBMC in the step (2) as a template;

(4) amplifying the variable region coding genes of the heavy chain and the light chain of the single B lymphocyte antibody by using the cDNA of the single B lymphocyte in the step (2) as a template;

(5) respectively and seamlessly splicing the antibody heavy chain variable region obtained in the step (4), the heavy chain constant region obtained in the step (3), the light chain variable region obtained in the step (4) and the light chain constant region sequence obtained in the step (3) by utilizing a fusion PCR technology to obtain a full-length heavy chain gene and a full-length light chain gene;

(6) inserting the full-length heavy chain gene obtained in the step (5) into the downstream of an EF-1 alpha promoter of a pBudCE4.1 eukaryotic expression vector, and simultaneously inserting the full-length light chain gene obtained in the step (5) into the downstream of a CMV promoter of the pBudCE4.1 eukaryotic expression vector;

(7) and (4) transfecting the swine-origin antibody eukaryotic expression vector obtained in the step (6) to 293T cells, collecting cell supernatants after transfection, screening the antibodies, and purifying the screened cell supernatants to obtain the full swine-origin African swine fever virus monoclonal antibody.

Further, the whole swine African swine fever virus monoclonal antibody heavy chain constant region is IgG1 type.

Further, the light chain constant region of the whole swine African swine fever virus monoclonal antibody is of Kappa type or Lambda type.

In the fifth aspect of the invention, the whole swine African swine fever virus monoclonal antibody can be used for preparing an African swine fever diagnostic reagent or an African swine fever treatment reagent.

The invention has the beneficial effects that:

the invention successfully obtains a full-swine-origin monoclonal antibody specific to African swine fever virus for the first time by utilizing a single B cell PCR technology. The antibody reserves natural pairing of light chain and heavy chain variable regions, has the advantages of good gene diversity, full swine origin, good antibody affinity, strong specificity and the like, can be efficiently expressed in a eukaryotic expression system, and can be used for screening antibody stable cell lines by utilizing carrier resistance in the later stage, thereby reducing the production cost. The invention provides a novel raw material for developing a diagnosis kit of African swine fever virus and also provides a new technical support for developing preventive and therapeutic drugs of the full-swine-derived monoclonal antibody.

Drawings

FIG. 1 shows the result of PCR amplification of genes encoding heavy and light chain constant regions;

m is DL2000 relative molecular mass standard; heavy chain constant region, size about 1000 bp; 2: a kappa light chain constant region of approximately 350bp in size; 3: lambda light chain constant region, approximately 350bp in size.

FIG. 2 shows the result of PCR amplification of a gene encoding a heavy chain variable region (HV);

m is DL2000 Marker; 1-5, different pores HV;

FIG. 3 shows the results of PCR amplification of the kappa and lambda light chain variable region encoding genes;

A. kappa light chain variable region (kappa V). M is DL2000 Marker; 1-5, different holes kappa V;

B. lambda light chain variable region (lambda V). M is DL2000 Marker; 1-5, different holes lambda V.

FIG. 4 shows the PCR splicing results of the full-length heavy and light chain fragments;

A. the heavy chain variable region and the constant region are spliced. M is DL2000 Marker; 1-2, the total length of the heavy chain is about 1500 bp;

B. kappa light chain variable and constant regions. M is DL2000 Marker; 1-2: kappa light chain full length, the size is about 750 bp;

C. the lambda light chain variable region and the constant region are spliced. M is DL2000 Marker; 1-2 lambda light chain total length, about 750 bp.

FIG. 5 shows Western blotting verification results of antibody eukaryotic expression vector transfected 293T cells;

A. identification of the kappa-9B antibody. M: a protein Marker; 1: 293T cell control transfected with empty vector; 2: transfecting a kappa-9B eukaryotic expression plasmid 293T cell;

B. Lambda-1A antibody identification results. M: a protein Marker; 1: 293T cell control transfected with empty vector; 3: lambda-1A eukaryotic expression plasmid 293T cells were transfected.

FIG. 6 shows the results of antibody purification;

m: a protein Marker; 1: kappa-9B purified antibody; 2: purified lambda-1A antibody.

Detailed Description

The present invention will be described in more detail with reference to the following embodiments for understanding the technical solutions of the present invention, but the present invention is not limited to the scope of the present invention.

1. Sample collection and PBMC isolation

The jugular vein was used to collect 10ml of anticoagulated blood from an African swine fever immune-tolerant pig (5 months old, from a pig farm in New county, Henan) for PBMC isolation. The operation method is the method described in the specification of a porcine peripheral blood lymphocyte separation kit (tertiary amine salt/TBD, LTS 1110).

2. Isolation and screening of target B lymphocytes

ASFV and anti-pig IgG double markers are adopted to select ASFV antigen specific pig B lymphocyte. After inactivation of ASFV, biotin (Thermo Scientific) is used^TMEZ-Link^TMSulfo-NHS-LC-Biotinylation Kit, Thermofish, 21435).

2.1 sorting of ASFV-positive B lymphocytes

The isolated lymphocytes were counted and diluted to 1X 10 with PBS⁷one/mL. mu.L of the cells were taken, 2. mu.L of anti-porcine IgG-Dylight650(Abcam, ab102133), biotin-labeled ASFV and streptavidin PE-Cy7(Invitrogen, 25-4317-82) bound to biotin were added to stain the cells, and the cell group and the single-stained group were used as controls, mixed and incubated at 4 ℃ for 30min in the dark. Centrifuging at 1000g/min for 5min, washing with PBS for 3 times, resuspending cells with 500 μ LPBS, and sorting B lymph by flow cytometryCells, single B lymphocytes double positive for Dylight650 and PE-Cy7 were isolated into wells of a 96-well plate.

2.2 treatment of Single B lymphocytes

Single Cell lysate (Thermo life Single Cell Lysis Kit, Thermofish, 4458235) was added to a 96-well plate in an amount of 10. mu.L/well.

3. Preparation of porcine ASFV antibody

The isolated PBMC or the sorted single B lymphocyte is first transcriptionally amplified to amplify the genes of the constant region and the variable region of the heavy chain and the light chain of the antibody. Sequencing and sequence comparison analysis are carried out on the amplification product, the full-length heavy chain and light chain genes are constructed by the functional amplification product through fusion PCR, then the genes are inserted into an eukaryotic expression vector, and the antibody is obtained through transfection of 293T cells for functional verification. The amplification of antibody variable region genes, full-length genes and construction of eukaryotic expression plasmids were performed essentially as described in the literature (Li, k., Bai, j., Du, l., Wang, x., Ke, c., Yan, w., Li, c., Ren, l., Han, h., Zhao, y.,2019.Generation of monoclonal antibodies based on single cell technologies.vector immunology and immunology 215,109913.).

3.1 amplification of antibody genes

3.1.1 Synthesis of cDNA

RNA from sorted PBMC or single B lymphocytes was used as a template, and was reverse-transcribed to synthesize cDNA using Oligo-dt or random primers (PrimeScript II 1st Strand cDNA Synthesis Kit, TAKARA, 6210A), respectively.

3.1.2 amplification of antibody constant region genes

Primers were designed based on the Genbank published porcine antibody heavy chain (Genbank No. U03781.1)/kappa (Genbank No. FP312898.2), lambda (Genbank No. CU467669.2) light chain constant region sequences. The amplification primers are as follows:

IgCH-F：5'-GCCCCCAAGACGGCCCCATCGGT-3'；

IgCH-R：5'-TCATTTACCCGGAGTCTGGGAG-3'；

IgCκ-F：5'-GGCTGATGCCAAGCCATCCGTC-3'；

IgCκ-R：5'-TCACACTCGTTCCTGCTGAAGCT-3'；

IgCλ-F：5'-GGTCAGCCCAAGGCCRCTCCC-3'；

IgCλ-R：5'-CTAGGCGCACTCGGAGGGCGTCA-3'；

wherein IgGH-F/IgCH-R is used for amplifying heavy chain constant region genes, IgCk-F/IgCk-R is used for amplifying kappa light chain constant region genes, and IgClambda-F/IgClambda-R is used for amplifying lambda light chain constant region genes. The template was 3.1.1 PBMC reverse transcription product. The PCR amplification system is as follows:

reagent material	Volume of
		2 XPCR buffer	25μL
dNTP Mixture	10μL
		cDNA	2μL
Upstream primer (10 pmol/. mu.L)	1μL
		Downstream primer (10 pmol/. mu.L)	1μL
DNA polymerase mixture	0.5μL
		DEPC water	11.5μL
Total volume	50μL

The PCR program for amplifying the heavy chain constant region is respectively as follows: 2min at 98 ℃,10 s at 98 ℃, 30s at 56 ℃, 1min at 72 ℃ and 30 cycles, and 10min at 72 ℃; the PCR program for amplifying the light chain constant region is respectively as follows: 2min at 98 ℃,10 s at 98 ℃, 30s at 55 ℃, 30s at 72 ℃ and 30 cycles. After the amplification is finished, 1% agarose gel electrophoresis is carried out, the size of the heavy chain constant region gene is about 1000bp, the size of the kappa/lambda light chain constant region gene is about 350bp, and the target fragment is recovered by cutting gel. The recovered target fragment is inserted into a pMD-19T vector, and the plasmid with correct sequencing verification is stored at-20 ℃ for later use.

3.1.3 amplification of antibody variable region genes

The variable region gene of the antibody is amplified by nested PCR. Firstly, single B cell cDNA obtained in 3.1.1 is used as a template, a first round of swine antibody IgG1 and kappa/lambda light chain primers are used for amplifying antibody variable region genes, and then a second round of swine antibody IgG1 and kappa/lambda light chain primers are used for amplifying antibody variable region genes by using a first round of products as a template. Antibody variable region gene two rounds of amplification primer references (Li, k., Bai, j., Du, l., Wang, x., Ke, c., Yan, w., Li, c., Ren, l., Han, h., Zhao, y.,2019.Generation of cyclic monoclonal antibody based on single cell technologies. vector immunology and immunology 215,109913.).

The PCR amplification system is as follows:

reagent material	Volume of
		2 XPCR buffer	25μL
dNTP Mixture	10μL
		cDNA	2μL
Upstream primer (10 pmol/. mu.L)	0.25. mu.L each
		Downstream primer (10 pmol/. mu.L)	0.25. mu.L each
DNA polymerase mixture	0.5μL
		DEPC water	Proper amount of water
Total volume	50μL

The PCR procedure for amplifying antibody variable regions was: 2min at 98 ℃,10 s at 98 ℃, 30s at 55 ℃, 30s at 72 ℃ and 30 cycles, and 10min at 72 ℃. The obtained product was subjected to 1% agarose gel electrophoresis, and PCR products (heavy chain variable region of about 400bp, kappa light chain variable region and lambda light chain variable region of about 350bp) conforming to the expected size were recovered and purified. And (5) sequencing and verifying gel recovery products.

3.1.4 amplification of the full-Length Gene of the antibody

The heavy/light chain constant regions and the heavy/light chain variable regions were used as templates, and the antibody heavy/light chains were ligated to the respective constant region fragments by fusion PCR, thereby obtaining the full-length heavy/light chain sequences. Leading peptide sequences are introduced into the upstream of the antibody gene, and enzyme cutting sites are introduced into the upstream and the downstream, so that the eukaryotic expression plasmid is conveniently constructed in the next step. The PCR amplification system is as follows:

the PCR procedure was: 2min at 98 ℃,10 s at 98 ℃, 30s at 55 ℃, 90s at 72 ℃ and 30 cycles, and 10min at 72 ℃. The obtained product was subjected to 1% agarose gel electrophoresis, and the PCR product (the total length of the heavy chain was about 1500bp, and the total length of the light chain was about 750bp) which corresponded to the expected size was recovered and purified.

3.1.5 construction of eukaryotic expression plasmids for antibodies

And (3) carrying out NotI/XhoI double enzyme digestion treatment on the antibody heavy chain full-length gel recovery product obtained in the step (3.1.4) and the pBudCE4.1 vector, and connecting after recovering the double enzyme digestion product gel, so that the antibody heavy chain full-length gene is inserted into the downstream of the EF-1 alpha promoter of the pBudCE4.1 vector. Then carrying out SalI/BamHI double enzyme digestion treatment on the plasmid and the recovered product of the antibody light chain full-length gel, aiming at inserting the light chain full-length gene into the downstream of a CMV promoter of a pBudCE4.1 vector, thereby obtaining a complete antibody eukaryotic expression plasmid.

3.1.6 antibody expression

293T cells were plated in 10cm dishes and transfected with 3.1.5 antibody expression plasmids after the cells reached approximately 80% of the monolayer. Mu.g of plasmid was added to 500. mu.L of Lopti-MEM, 10. mu.L of transfection reagent lipofectamine 2000(Invitrogen, 11668-019) was added thereto, the mixture was blown to mix well, the mixture was allowed to react at room temperature for 15min, and 293T cells were added thereto. Cells and supernatant were collected for antibody functional validation 72h after transfection. Obtaining two pig source monoclonal antibodies which can identify African swine fever and are named as kappa-9B and lambda-1A, wherein the amino acid sequences of the heavy chain constant regions of the kappa-9B antibodies are shown as SEQ ID NO. 9, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, the amino acid sequence of the kappa type light chain constant region is shown as SEQ ID NO. 10, and the amino acid sequence of the kappa type light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain constant region of the lambda-1A antibody is shown as SEQ ID NO. 9, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5, the amino acid sequence of the lambda type light chain constant region is shown as SEQ ID NO. 11, and the amino acid sequence of the lambda type light chain variable region is shown as SEQ ID NO. 6.

3.1.7 purification of antibodies

The cell supernatants collected at 3.1.6 were purified by antibody, according to the instructions of HiTrap Protein A HP from GE Healthcare.

4. Antibody identification and screening

4.1 Western blotting detection method for swine antibodies

After antibody transfection of 293T cells, the cells were lysed with RIPA lysate, cells transfected with empty vector served as control, and after cell lysis 5X protein loading buffer was added and boiled in boiling water for 10 min. The samples were run on SDS-PAGE gels and after running the gels were transferred to nitrocellulose membranes (NC). The NC membrane was blocked in 5% skimmed milk powder for 1h at room temperature, and then HRP-pig IgG diluted with 0.05% PBST (1 ten thousand fold dilution) was added, the membrane was washed 4 times for 5 min/time at room temperature for 1h, 0.05% PBST. ECL developer was then added and developed on a ChemiDocTM XRS chemiluminescent imaging analysis system.

4.2 characterization of expressed antibody Activity

4.2.1 coating ASFV detection antibody

Diluting ASFV inactivated antigen with 50mM carbonate buffer solution (pH 9.6) at a ratio of 1:1 to coat the ELISA plate, coating at 50 μ L/well, and coating overnight at 4 ℃; washing with 0.05% PBST washing solution for 4 times, and drying; adding 100 μ L trehalose (8.4g/L), sealing at 4 deg.C for 10 hr; spin-drying, adding antibody transfection supernatant or purified antibody (0.1mg/mL)50 μ L/hole, and incubating at 37 deg.C for 30 min; washing with 0.05% PBST washing solution for 4 times, and drying; diluting the HRP-labeled porcine secondary antibody by 1:20000, then performing 50 mu L/hole reaction for 30min at 37 ℃; washing with 0.05% PBST washing solution for 4 times, and drying; adding 50 μ L TMB substrate solution, incubating at 37 deg.C for 15min, adding 50 μ L stop solution (2M H)₂SO₄) Reading the OD of the reaction on a spectrophotometer_450nmThe value is obtained.

4.2.2 detection of ASFV by coated antibody

The purified antibody is diluted to 0.1mg/ml with 50mM carbonate buffer (pH 9.6)ml coated enzyme label plate, 50 μ L/hole, 4 ℃ overnight coating; washing with 0.05% PBST washing solution for 4 times, and drying; adding 100 μ L trehalose (8.4g/L), sealing at 4 deg.C for 10 hr; after the sealing is finished, adding 50 mu L of ASFV antigen, and performing incubator action at 37 ℃ for 30 min; washing with 0.05% PBST washing solution for 4 times, and drying; adding 50 μ L of HRP-labeled secondary pig antibody 1 diluted with 20000, incubating at 37 deg.C for 30min, washing with 0.05% PBST for 4 times, and drying; adding 50 μ L TMB substrate solution, incubating at 37 deg.C for 15min, adding 50 μ L stop solution (2M H)₂SO₄) Reading the OD of the reaction on a spectrophotometer_450nmThe value is obtained.

5. Results

5.1 amplification of genes encoding heavy/light chain constant regions

The PBMC cDNA was used as a template to amplify the constant regions of the heavy chain and kappa/lambda light chain of the antibody, and the PCR product was identified by 1% agarose gel electrophoresis, as shown in FIG. 1, the fragments of the constant region of the heavy chain of the antibody were about 1000bp, and the fragments of the constant region of the kappa/lambda light chain of the antibody were about 350 bp.

5.2 amplification of genes encoding the variable region of the heavy chain

Amplifying single B lymphocyte cDNA sorted from 96 holes to obtain a PCR product with the size of about 400bp, wherein the PCR product is consistent with the size of an expected amplification product; after the gel is cut and recovered, the PCR product is sent for sequencing. The sequencing result is compared with an antibody gene library (IMGT) for analysis, and the sequence obtained by amplification is confirmed to be the swine African swine fever virus monoclonal antibody heavy chain variable region (HV), as shown in figure 2.

5.3 amplification of genes encoding the light chain variable region

Amplifying single B lymphocyte cDNA sorted from 96 holes to obtain a PCR product with the size of about 350bp, wherein the PCR product is consistent with the size of an expected amplification product; after the gel is cut and recovered, the PCR product is sent for sequencing. The sequencing result and the antibody gene library (IMGT) are compared and analyzed, and the sequences obtained by amplification are proved to be the kappa light chain (kappa V) and the lambda light chain variable region (lambda V) of the swine African swine fever virus monoclonal antibody, as shown in figure 3.

5.4 splicing of full-Length fragments of antibodies

The antibody variable region and constant region fragments were seamlessly joined using fusion PCR to assemble full-length fragments of heavy and light chains. The size of the whole length of the heavy chain is about 1500 bp; the full length of lambda and kappa chains is about 750 bp; consistent with the expected amplification results, as shown in fig. 4.

5.5 Western blotting detection of antibody transfected cells

In order to further verify that the obtained antibody is a swine antibody, the transfected antibody and the empty vector 293T cell are respectively collected and detected by using a swine HRP-IgG antibody, and Western blotting results show that a specific band can be detected in the transfected antibody group, but no band is detected in the empty vector control group, which shows that the obtained antibody is a swine antibody, as shown in FIG. 5.

5.6 ELISA detection of antibody transfection supernatants

After the antibody eukaryotic expression plasmid is transfected into 293T cells, ELISA detection is carried out on the supernatant, the cell supernatant transfected with an empty vector is used as a negative control, as shown in Table 1, the OD value of an antibody transfection group is obviously higher than that of the negative control, which indicates that the antibody eukaryotic expression plasmid can secrete an antibody after being transfected into the 293T cells, and the antibody can be combined with ASFV, and is proved to be a swine anti-ASFV antibody.

TABLE 1 ELISA test results of antibody transfection supernatants

Sample (I)	OD value
		κ-9B	0.733
λ-1A	0.873
		Negative control	0.242

5.7 purification of antibodies

The collected supernatant of the transfected antibody cells was combined with Protein A, and further porcine IgG was isolated. The eluted product is identified by SDS-PAGE, and the result shows that the purer pig-derived monoclonal antibody is obtained, as shown in FIG. 6.

5.8 ELISA detection of purified antibody

5.8.1 ASFV coating detection of antibody Activity

ASFV antigen is coated on an enzyme label plate, detection is carried out according to the procedure of 4.2.1, the detection result is shown in Table 2, and the result shows that the ASFV is specifically combined with porcine antibodies kappa-9B and lambda-1A.

TABLE 2 purified antibody ELISA test results

Sample (I)	OD value
		κ-9B	1.908
λ-1A	2.603
		Negative control	0.250

2.8.2 detection of ASFV by coating with purified antibody

The purified antibodies were coated on an ELISA plate and detected according to the procedure described in 4.2.2, and the results are shown in Table 3, which indicates that the purified kappa-9B and lambda-1A antibodies can specifically bind to ASFV.

TABLE 3 ASFV ELISA test results

Sample (I)	OD value
		κ-9B	0.717
λ-1A	0.873
		Negative control	0.199

The above-described embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications in the structure, features and principles described in the present invention should be included in the claims of the present invention.

SEQUENCE LISTING

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> full-swine-origin African swine fever virus monoclonal antibody and preparation method thereof

<130> do not

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Ala Met Ser Asp Ser Gly Gly Arg Asn Cys Val Ala Gly Asp Ser Ser

100 105 110

Cys Pro His Tyr Tyr Gly Met Asn Leu Trp Gly Pro Gly Val Glu Val

115 120 125

Val Val Ser

130

<210> 6

<211> 108

<212> PRT

<213> African swine fever virus

<400> 6

Ser Gln Thr Val Ile Gln Glu Pro Ala Met Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Ala Phe Ser Ser Gly Ser Val Thr Thr Ser

20 25 30

Asn Tyr Pro Gly Trp Tyr Gln Gln Leu Pro Gly Gln Pro Pro Arg His

35 40 45

Leu Ile Gly Gly Thr Asn Ser Arg Pro Thr Gly Val Pro Gly Arg Phe

50 55 60

Ser Gly Ala Ile Ser Gly Asn Lys Ala Thr Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Leu Tyr Lys Ser Pro

85 90 95

Thr Ile Phe Gly Gly Gly Thr His Leu Thr Val Leu

100 105

<210> 7

<211> 396

<212> DNA

<213> African swine fever virus

<400> 7

gaggggcagc tggtggagtc tggaggaggc ctggtgcagc ctggggggag tctgagactc 60

tcctgtgtcg gctctggatt cgacttcata accgcctaca tgaactgggt ccgccaggct 120

ccagggaagg ggctggagtg gctggccacc attagcacta agggtattag tacttactat 180

gcagactccg tgaagggccg attcaccatc tccaaagaca actccaagag cacggccgat 240

ctgctcatgc gcagcctgag aatcgaagac acggcccgct atttctgtgc aatgagcgat 300

agcggtggtc gaaattgcgt tgccggggat tcttcgtgtc cccattacta tggtatgaat 360

ctctggggcc caggcgttga agtcgtcgtg tcctca 396

<210> 8

<211> 324

<212> DNA

<213> African swine fever virus

<400> 8

tctcagactg tgatccagga gccggcgatg tcagtgtctc ctggagggac cgtcacactc 60

acctgtgcct ttagctctgg gtcagtcact actagtaact accccggctg gtaccagcag 120

ctaccaggcc agcctccccg acacctgatc ggcggcacaa acagccgccc gactggggtc 180

cccggtcgct tctctggagc catctctggg aacaaagcca ccctcaccat cacgggggcc 240

caggctgagg acgaggccga ctacttttgt gctctctata aaagccctac tattttcggc 300

ggtgggaccc atctgaccgt cctc 324

<210> 9

<211> 328

<212> PRT

<213> African swine fever virus

<400> 9

Ala Pro Lys Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Cys Gly Arg

1 5 10 15

Asp Thr Ser Gly Pro Asn Val Ala Leu Gly Cys Leu Ala Ser Ser Tyr

20 25 30

Phe Pro Glu Pro Val Thr Met Thr Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ser Val Leu Gln Pro Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Met Val Thr Val Pro Ala Ser Ser Leu Ser Ser Lys Ser

65 70 75 80

Tyr Thr Cys Asn Val Asn His Pro Ala Thr Thr Thr Lys Val Asp Lys

85 90 95

Arg Val Gly Thr Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys

100 105 110

Glu Val Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Thr Ala Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gln His Gln Asp Trp

180 185 190

Leu Lys Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Val Asp Leu Pro

195 200 205

Ala Pro Ile Thr Arg Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser

225 230 235 240

Lys Val Thr Val Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile

245 250 255

His Val Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr

260 265 270

Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr Phe Phe Leu Tyr

275 280 285

Ser Lys Leu Ala Val Asp Lys Ala Arg Trp Asp His Gly Glu Thr Phe

290 295 300

Glu Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

305 310 315 320

Ser Ile Ser Lys Thr Gln Gly Lys

325

<210> 10

<211> 108

<212> PRT

<213> African swine fever virus

<400> 10

Ala Asp Ala Lys Pro Ser Val Phe Ile Phe Pro Pro Ser Lys Glu Gln

1 5 10 15

Leu Glu Thr Gln Thr Val Ser Val Val Cys Leu Leu Asn Ser Phe Phe

20 25 30

Pro Arg Glu Val Asn Val Lys Trp Lys Val Asp Gly Val Val Gln Ser

35 40 45

Ser Gly Ile Leu Asp Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Ser Leu Pro Thr Ser Gln Tyr Leu Ser

65 70 75 80

His Asn Leu Tyr Ser Cys Glu Val Thr His Lys Thr Leu Ala Ser Pro

85 90 95

Leu Val Lys Ser Phe Ser Arg Asn Glu Cys Glu Ala

100 105

<210> 11

<211> 106

<212> PRT

<213> African swine fever virus

<400> 11

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

1 5 10 15

Glu Glu Leu Gly Thr Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Thr Trp Lys Ala Gly Gly Thr Thr

35 40 45

Val Thr Gln Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ala Leu Ser Ala Ser Asp Trp Lys

65 70 75 80

Ser Ser Ser Gly Phe Thr Cys Gln Val Thr His Glu Gly Thr Ile Val

85 90 95

Glu Lys Thr Val Thr Pro Ser Glu Cys Ala

100 105

Claims

1. The monoclonal antibody of the African swine fever virus of the whole pig source is characterized by comprising a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO 1 or SEQ ID NO 5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO 2 or SEQ ID NO 6.

2. The monoclonal antibody against African swine fever virus of whole pig origin according to claim 1, wherein the heavy chain constant region is of type IgG 1.

3. The monoclonal antibody against African swine fever virus of whole pig origin according to claim 1, wherein the light chain constant region is Kappa-type or Lambda-type.

4. A DNA fragment encoding the monoclonal antibody against African swine fever virus of all porcine origin according to claim 1.

5. The DNA fragment encoding the monoclonal antibody against swine fever virus, according to claim 4, wherein the DNA fragment encoding the heavy chain variable region of the monoclonal antibody against swine fever virus is represented by SEQ ID NO. 3 or SEQ ID NO. 7, and the DNA fragment encoding the light chain variable region of the monoclonal antibody against swine fever virus is represented by SEQ ID NO. 4 or SEQ ID NO. 8.

6. A recombinant expression vector comprising the DNA fragment of any one of claims 4 to 5.

7. A host cell comprising the recombinant expression vector of claim 6.

8. The use of the monoclonal antibody against African swine fever virus derived from swine of claim 1 in the preparation of a diagnostic reagent or a therapeutic reagent for African swine fever.