CN113150124B

CN113150124B - Double-antibody sandwich ELISA based on African swine fever virus p72 gene and application thereof

Info

Publication number: CN113150124B
Application number: CN202110084466.4A
Authority: CN
Inventors: 冯春燕; 林祥梅; 王彩霞; 吴绍强; 刘晓飞; 仇松寅
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2021-01-21
Filing date: 2021-01-21
Publication date: 2022-07-29
Anticipated expiration: 2041-01-21
Also published as: CN113150124A

Abstract

The invention discloses an antigen binding protein, an antibody or an active fragment specifically binding to African swine fever virus p72 protein, a double-antibody sandwich ELISA antigen detection kit containing the African swine fever virus p72 protein of the antigen binding protein, the antibody or the active fragment, and application thereof. The kit comprises an ELISA plate coated with a monoclonal antibody ASFV-p72-5G8, a positive control, a negative control, an HRP-marked detection antibody ASFV-p72-12F6, a sample diluent, a developing solution and a washing solution. The antibodies ASFV-p72-5G8 and ASFV-p72-12F6 are secreted by hybridoma cell strains ASFV-p72-5G8 and ASFV-p72-12F6 respectively. The kit of the invention establishes a double-antibody sandwich ELISA method by taking 5G8 as a capture antibody and 12F6 coupled HRP as a detection antibody, can specifically detect ASFV, has simple and convenient operation, high sensitivity and good specificity, and provides a new detection means for clinical diagnosis and epidemiological investigation of ASFV infection.

Description

Double-antibody sandwich ELISA based on African swine fever virus p72 gene and application thereof

Technical Field

The invention belongs to the field of biotechnology detection, and particularly relates to a double-antibody sandwich ELISA antigen detection kit based on African swine fever virus p72 gene and application thereof in fresh blood detection of sick animals.

Background

African Swine Fever (ASFV) is an acute, virulent and highly contagious disease of pigs caused by African Swine Fever Virus (ASFV), and clinical symptoms of the ASFV are highly similar to that of Classical Swine Fever (CSF), and the ASFV is also originated from African swine fever, so the ASFV is called African swine fever.

Along with the development of monoclonal antibody technology in recent years, the research aiming at African swine fever monoclonal antibody plays more and more prominent roles in ASFV research and ASFV diagnosis, which is mainly shown in two aspects, namely, the monoclonal antibody is used for establishing an immunology-related ASFV detection method, such as direct immunofluorescence assay (DIF) for detecting ASFV antigen, an immunoenzyme histochemical assay, a sandwich enzyme-linked immunosorbent assay and the like; the second is used for the research of virus and antigen structure and the research of ASFV antigen characteristic. Although no ASFV vaccine exists at present, the ASFV antibody is positive, which indicates that the infection is present or once, but does not represent that no ASFV vaccine exists in the future, so the detection of the ASFV antigen is particularly important, and epidemic prevention and control measures can be taken only by accurately determining the infection condition.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an antigen binding protein, an antibody or an active fragment specifically binding to African swine fever virus p72 protein, and a double-antibody sandwich ELISA kit based on the African swine fever virus p72 gene, wherein the kit can be used for rapidly and accurately detecting ASFV antigen.

In a first aspect, the invention provides an artificially modified p72 protein, wherein the p72 protein has a gene sequence shown in SEQ ID NO. 31.

In a second aspect, the present invention provides an antigen binding protein that specifically binds to the p72 protein, the antigen binding protein having the heavy chain complementarity determining region CDR1 shown in SEQ ID NO:19, the heavy chain complementarity determining region CDR2 shown in SEQ ID NO:20, the heavy chain complementarity determining region CDR3 shown in SEQ ID NO:21, and the light chain complementarity determining region CDR1 shown in SEQ ID NO:22, the light chain complementarity determining region CDR2 shown in SEQ ID NO:23, and the light chain complementarity determining region CDR3 shown in SEQ ID NO: 24.

Preferably, the antigen binding protein comprises at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 1, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 2, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

Wherein the content of the first and second substances,

the amino acid sequence shown in SEQ ID NO. 1 is as follows:

EVQLQQSGAELVKPGASVKLSCTASGFNASIKDTYMHWVKQRPEQGLAWIGMIDPA NCTGNSKFDPKFQGKATIAADTSSNTAYLQLSSLTSEDTAVYYCGGLSDTYYGKYEGDYY GLDCWGQGTSVTVSS；

the amino acid sequence shown in SEQ ID NO. 2 is as follows:

DIVMSQSPYSPAVSVGEKVTMSCKSSQSLLYESYSQKNYLAWYQQKPGQSPKLLIYW AVPSTRESGVPDRFTGSGSGTDFTLTISSVRAEDLAIYYCHQYYTSVYPWTFGGGTKLEIK。

in a third aspect, the present invention provides an antigen binding protein that specifically binds to the p72 protein, the antigen binding protein having the heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 25, the heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 26, the heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 27, and the light chain complementarity determining region CDR1 shown in SEQ ID NO. 28, the light chain complementarity determining region CDR2 shown in SEQ ID NO. 29, and the light chain complementarity determining region CDR3 shown in SEQ ID NO. 30.

Preferably, the antigen binding protein comprises at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 3, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 4, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 4.

Wherein the content of the first and second substances,

the amino acid sequence shown in SEQ ID NO. 3 is as follows:

QIQLVQSGPELKKPGETVRISCKASGYISMFTTAGMEWVQKKPGKGLKWIGWINTHS LQGVTKNGEDFKGRFAFSLETSASTTYLQISNLKNEDTATYFCARWGNYHNADGMDYW GQGTSVTVSS；

The amino acid sequence shown in SEQ ID NO. 4 is as follows:

DIQMNQSPSSLSASLGDTITITCHASQNINDTVWLSWYQQKPGNIPKLLIYRTSQLNL HTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGTPQSYPLTFGGGTKLEIK。

in a fourth aspect, the present invention provides an antibody or active fragment that specifically binds to the p72 protein, the antibody or active fragment having the heavy chain complementarity determining region CDR1 shown in SEQ ID NO:19, the heavy chain complementarity determining region CDR2 shown in SEQ ID NO:20, the heavy chain complementarity determining region CDR3 shown in SEQ ID NO:21, and the light chain complementarity determining region CDR1 shown in SEQ ID NO:22, the light chain complementarity determining region CDR2 shown in SEQ ID NO:23, and the light chain complementarity determining region CDR3 shown in SEQ ID NO: 24.

Preferably, the antibody or active fragment comprises at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 1, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 2, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

More preferably, the antibody or active fragment is a monoclonal antibody and/or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a single-chain antibody fragment, a chimeric monoclonal antibody fragment, a modified monoclonal antibody and a modified monoclonal antibody fragment; further preferably, the antibody is a murine monoclonal antibody ASFV-p72-5G 8.

In a preferred embodiment, the antibody ASFV-p72-5G8 is prepared by the following method: immunizing a mouse by using p72 protein expressed by a recombinant eukaryotic system, taking spleen cells of the immunized mouse, fusing with SP2/0 cells, screening to obtain monoclonal hybridoma cells 5G8 secreting specific antibodies, injecting the hybridoma cells into the abdominal cavity of the mouse, collecting ascites after the abdominal cavity of the mouse is swelled, and obtaining the monoclonal antibody ASFV-p72-5G8 containing the African swine fever virus by carrying out salt purification on the ascites.

In a fifth aspect, the present invention provides an antibody or an active fragment that specifically binds to the p72 protein, the antibody or the active fragment having the heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 25, the heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 26, the heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 27, and the light chain complementarity determining region CDR1 shown in SEQ ID NO. 28, the light chain complementarity determining region CDR2 shown in SEQ ID NO. 29, and the light chain complementarity determining region CDR3 shown in SEQ ID NO. 30.

Preferably, the antibody or active fragment comprises at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 3, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids of the amino acid sequence shown in SEQ ID NO. 3; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 4, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 4.

More preferably, the antibody or active fragment is a monoclonal antibody and/or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a single-chain antibody fragment, a chimeric monoclonal antibody fragment, a modified monoclonal antibody and a modified monoclonal antibody fragment; further preferably, the antibody is a murine monoclonal antibody ASFV-p72-12F 6.

In a preferred embodiment, the antibody ASFV-p72-12F6 is prepared by the following method: immunizing a mouse by using p72 protein expressed by a recombinant eukaryotic system, taking spleen cells of the immunized mouse, fusing with SP2/0 cells, screening to obtain monoclonal hybridoma cells 12F6 secreting specific antibodies, injecting the hybridoma cells into the abdominal cavity of the mouse, collecting ascites after the abdominal cavity of the mouse is swelled, and obtaining the monoclonal antibody ASFV-p72-12F6 containing the African swine fever virus by carrying out salt purification on the ascites.

In a sixth aspect, the invention provides an African swine fever virus p72 gene double-antibody sandwich ELISA antigen detection kit, which comprises the antigen binding protein, antibody or active fragment specifically binding to the African swine fever virus p72 protein.

Preferably, the antibodies are murine monoclonal antibodies ASFV-p72-5G8 and ASFV-p72-12F 6.

In a preferred embodiment, the double antibody sandwich ELISA antigen detection kit of the present invention comprises: an ELISA plate coated with a monoclonal antibody ASFV-p72-5G8, a positive control, a negative control, an HRP-marked detection antibody ASFV-p72-12F6, a sample diluent, a developing solution and a washing solution.

In a seventh aspect, the invention provides an antigen binding protein, an antibody or an active fragment thereof, which specifically binds to the African swine fever virus p72 protein, or an application of the double-antibody sandwich ELISA antigen detection kit in detection of African swine fever virus in a sample.

Preferably, the sample is a serum sample or a blood sample.

In an eighth aspect, the invention provides a method for in vitro detection of African swine fever virus in a sample by using an antigen binding protein, antibody or active fragment that specifically binds to the p72 protein of African swine fever virus, or the double-antibody sandwich ELISA antigen detection kit.

Preferably, the sample is a serum sample or a blood sample.

The invention has the beneficial effects that:

(1) the invention uses two auxiliary proteins B602L and B438L to assist the expression of the p72 protein in eukaryotic cells, so that the space structure of the natural virus antigen is easier to form in the expression process, the antigen of the monoclonal antibody prepared by the invention is closer to the real virus antigen, and the prepared antigen has high purity and good immunogenicity and antigenicity.

(2) The monoclonal antibody aiming at the ASFV p72 protein has high titer and stable property, can prepare the antibody by using mouse ascites, and is suitable for mass production.

(3) The invention uses high-purity eukaryotic expressed ASFV p72 protein as immunogen and detecting antigen to prepare the monoclonal antibody of ASFV p72 protein, and two strains (5G8 and 12F6) are selected from the obtained monoclonal antibody to establish a double-antibody sandwich ELISA method for detecting ASFV antigen, which can detect ASFV antigen rapidly and accurately.

(4) According to the invention, 8 monoclonal antibodies are obtained in the process of preparing the ASFV p72 protein specific monoclonal antibody, 2 pairs of monoclonal antibodies 5G8 and 12F6 with good effect are finally screened through antigen site analysis and pairing test, the subclass identification is determined to be an IgG1 subtype, and light chains are all Kappa subtypes. The hybridoma cells ASFV-p72-5G8 and the monoclonal antibodies 5G8 and 12F6 secreted by ASFV-p72-12F6 can generate specific reaction with ASFV infected cells, the two cells have stable biological characteristics, and the secreted monoclonal antibodies are directed at different epitopes of ASFV p72 protein, thereby having important application value in ASFV infection diagnosis. The invention relates to a double-antibody sandwich ELISA method established by taking a monoclonal antibody 5G8 secreted by a hybridoma cell strain ASFV-p72-5G8 as a capture antibody and a monoclonal antibody 12F6(HRP coupling) secreted by a hybridoma cell strain ASFV-p72-12F6 as a detection antibody, which can specifically detect ASFV p72 protein, and the method has higher linear coefficient and wider detection range, high sensitivity and good specificity.

According to the present invention, certain amino acids in the amino acid sequence can be conservatively substituted without altering the activity or function of the protein, see table a below:

TABLE A

Residue of	Conservative substitutions	Residue of	Conservative substitutions
				Ala	Ser	Leu	Ile；Val
Arg	Lys	Lys	Arg；Gln
				Asn	Gln；His	Met	Leu；Ile
Asp	Glu	Phe	Met；Leu；Tyr
				Gln	Asn	Ser	Thr；Gly
Cys	Ser	Thr	Ser；Val
				Glu	Asp	Trp	Tyr
Gly	Pro	Tyr	Trp；Phe
				His	Asn；Gln	Val	Ile；Leu
Ile	Leu；Val

Furthermore, because of the degeneracy of the bases, substitutions can be made to bases of a polynucleotide sequence without altering the activity or function of the polynucleotide sequence, see table B below:

TABLE B

Drawings

FIG. 1 is the peak elution and SDS-PAGE identification of p72 recombinant protein.

FIG. 2 shows the result of Western-blot analysis of p72 recombinant protein.

1: a protein Marker; 2: purified recombinant p72 protein.

FIG. 3 is the identification of the specificity of the recombinant p72 protein.

1: a protein Marker; 2: standard positive sera; 3: porcine reproductive and respiratory syndrome virus (HuN4-F122 strain) positive sera; 4: porcine parvovirus (WH-1) positive serum; 5: hog cholera virus (strain C) positive serum; 6: porcine circovirus type 2 (LG strain) positive serum; 7: porcine pseudorabies virus (Kartha-K61 strain) positive sera; 8: pig negative serum.

FIG. 4 is an immunogenicity analysis of recombinant p72 protein.

FIG. 5 shows the results of indirect immunofluorescence assay of 8 monoclonal antibody and baculovirus-expressed p 72.

FIG. 6 is a schematic representation of SDS-PAGE detection of purified monoclonal antibodies (5G 8).

FIG. 7 shows the result of chromosome detection of hybridoma cells.

FIG. 8 shows the results of the monoclonal antibody subclass measurement.

FIG. 9 is a standard curve and regression analysis of p72 protein quantitative determination.

Detailed Description

In order to quickly and accurately diagnose ASFV infection, the invention develops the monoclonal antibody which can have good reactivity with ASFV p72 protein, and further prepares the double-antibody sandwich ELISA detection kit for detecting p72 protein. The present invention will be further specifically described below by way of specific examples, but it should not be construed as limiting the scope of the present invention.

Example 1 preparation and characterization of recombinant protein of African swine fever virus p72

1. Material

1.1 bacterial species

Coli competence DH5 alpha, DH10Bac were purchased from Beijing holotype gold biotechnology, Inc., pCMV vector, pFastbac1 vector, 293F cells were preserved by animal inspection and quarantine laboratory of Chinese inspection and quarantine science research institute.

1.2 serum

Standard positive sera for african swine fever inactivated with 0.3% TNBP and 1% triton X-100 were preserved by the national veterinary institute of polish. Porcine Pseudorabies live vaccine (Kartha-K61 strain) (PRV) purchased from Qingdao Yibang bioengineering Co., Ltd; porcine parvovirus inactivated vaccine (WH-1) (PPV), purchased from Pre-biological GmbH, Wuhan's family; porcine circovirus type 2 inactivated vaccine (LG strain) (PCV 2) was purchased from harbin victoriaceae biotechnology development company, Swine Fever live vaccine (C strain) (classic Swine river, CSF), highly pathogenic Porcine reproductive and respiratory syndrome live vaccine (HuN4-F122 strain) (Porcine reproductive and respiratory syndrome, PRRS) was purchased from harbin group biological vaccine ltd. Porcine negative control serum was purchased from Hyclone.

1.3 reagent

The SMM 293-TI medium is purchased from Beijing Yiqiao Shenzhou biotechnology, and the Ni-NTA agarose gel resin and the plasmid extraction kit are purchased from QIAGEN; the BCA protein concentration determination kit is purchased from Beijing Solaibao science and technology Limited; RPMI1640 medium and fetal bovine serum were purchased from Gibico; EcoR I/Xho I restriction enzyme, T4 ligase, TaKaRa LA PCR ^TM Kit was purchased from TaKaRa, DNA extraction Kit DNeasy Blood and Tissue Kit was purchased from Qiagen, Agarose Gel DNA Purification Kit, Plasmid Mini Kit was purchased from Omega, IPTG, X-gal, ampicillin were purchased from Beijing Quanjin Biotechnology Ltd.

1.4 instruments

General PCR instruments (AB Applied Biosystems) from ABI, bench centrifuges (3-18k) from sigma, constant temperature shaking incubator (HZQ-F100), fluorescence microscope (ZEISS, AXIO), six-electrophoresis apparatus, and double infrared laser scanning imaging system (Proteinimple).

2. Expression of recombinant p72 protein

2.1 construction of the p72 recombinant plasmid

According to the full-length sequence (1941bp) of the p72 gene of African swine fever Heilongjiang strain (MK333180.1) published in GenBank, physical and chemical properties, secondary structure and tertiary structure analysis of p72 protein and Prediction of T, B lymphocyte epitope are carried out by utilizing bioinformatics technologies of ExPASy, SOMPA, PSIPRED Server, DNASTAR, Phyre, ABCcred Prediction, Scratch, Nectcle, IEDB and the like, and finally, an N-terminal dominant antigen epitope region is selected to be linked by a Linker and a loop functional region at 462-545 bit is folded to form 3 repeated sequences, wherein the specific sequences are as follows:

atggcatcaggaggagctttttgtcttattgctaacgatgggaaggccgacaagattatattggcccaagacttgctgaatagcaggatc tctaacattaaaaatgtgaacaaaagttatgggaaacccgatcccgaacccactttgagtcaaatcgaagaaacacatttggtgcattttaatgc gcattttaagccttatgttccagtagggtttgaatacaataaagtacgcccgcatacgggtacccccaccttgggaaacaagcttacctttggtat tccccagtacggagactttttccatgatatggtgggccatcatatattgggtgcatgtcattcatcctggcaggatgctccgattcagggcacgtc ccagatgggggcccatgggcagcttcaaacgtttcctcgcaacggatatgactgggacaaccaaacacccttagagggcgccgtttacacg cttgtagatccttttggaagacccattgtacccggcacaaagaatgcgtaccgaaacttggtttactactgcgaataccccggagaacgacttta tgaaaacgtaagattcgatgtaaatggaaattccctagacgaatatagttcggatgtcacaacgcttgtgcgcaaattttgcatcccaggggata aaatgactggatataagcacttggttggccaggaggtatcggtggagggaaccagtggccctctcctatgcaacattcatgatttgcacaagc cgcaccaaagcaaacctattcttaccgatgaaaatgatacgcagcgaacgtgtagccataccaacccgaaatttctttcacagcattttcccga gaactctcacaatatccaaacagcaggtaaacaagatattactcctatcacggacgcaacgtatctggacataagacgtaatgttcattacagct gtaatggacctcaaacccctaaatactatcagccccctcttgcgctctggattaagttgcgcttttggtttaatgagaacgtgaaccttgctattcc ctcagtatccattcccttcggcgagcgctttatcaccataaagcttgcatcgcaaaaggatttggtgaatgaatttcctggactttttgtacgccag tcacgttttatagctggacgccccagtagacgcaatatacgctttaaaccatggtttatcccaggagtcattaatgaaatctcgctcacgaataat gaactttacatcaataacctgtttgtaacccctgaaatacacaacctttttgtaaaacgcGGTGGTGGTGGTGGTGGTTCCcc cattgaatatatgtttataggattaaaacctacctggaacatctccgatcaaaatcctcatcaacaccgagattggcacaagttcggacatgttgtt aacgccattatgcagcccactcaccacgcagagataagctttcaggatagagatacagctcttccagacgcatgttcatctatatctgatattag ccccgttacgtatccgatcacattacctattattaaaaacatttccgtaactgctcatGGTGGTGGTGGTGGTGGTTCCcccat tgaatatatgtttataggattaaaacctacctggaacatctccgatcaaaatcctcatcaacaccgagattggcacaagttcggacatgttgttaa cgccattatgcagcccactcaccacgcagagataagctttcaggatagagatacagctcttccagacgcatgttcatctatatctgatattagcc ccgttacgtatccgatcacattacctattattaaaaacatttccgtaactgctcatGGTGGTGGTGGTGGTGGTTCCcccattg aatatatgtttataggattaaaacctacctggaacatctccgatcaaaatcctcatcaacaccgagattggcacaagttcggacatgttgttaacg ccattatgcagcccactcaccacgcagagataagctttcaggatagagatacagctcttccagacgcatgttcatctatatctgatattagcccc gttacgtatccgatcacattacctattattaaaaacatttccgtaactgctcat(SEQ ID NO:31)。

The gene sequence is inserted into a eukaryotic expression vector to prepare an antigen, and the monoclonal antibody is obtained.

The gene sequence is artificially synthesized by Beijing Okagaku Biotechnology Co., Ltd, EcoR I enzyme cutting sites and Xho I enzyme cutting sites are respectively introduced at two ends of the sequence, 10 His and 3 Flag B646L sequences are introduced at the N end of the sequence through the two enzyme cutting sites and are inserted into a pCMV vector to construct a pCMV-ASFV-p72 recombinant plasmid, escherichia coli DH5 alpha competent cells are transformed, and the plate coating is carried out for overnight culture. And (4) selecting positive clones on the next day for sequencing, adding glycerol into positive bacteria liquid with correct sequencing for bacteria preservation, and preserving at-80 ℃ for later use.

2.2 construction of B602L and B438L recombinant plasmids

According to the B602L gene full-length sequence (1953bp) of African swine fever Heilongjiang strain (MK333180.1) published in GenBank, the sequence is synthesized by Beijing engine science and technology Limited, EcoR I enzyme cutting sites and Xho I enzyme cutting sites are respectively introduced at two ends of the sequence, B602L target gene sequences of 1 Flag and 1 Strep introduced at the N end are inserted into a pCMV vector through the two enzyme cutting sites to construct a pCMV-ASFV-B602L recombinant plasmid, Escherichia coli DH5 alpha competent cells are transformed, and the pCMV vector is plated for overnight culture. And (4) selecting positive clones for sequencing on the next day, adding glycerol into positive bacteria liquid with correct sequencing for bacteria preservation, and storing at-80 ℃ for later use.

According to the full-length sequence (1317bp) of the B438L gene of an African swine fever Heilongjiang strain (MK333180.1) published in GenBank, the gene is synthesized by Beijing Openkefamily Biotechnology Limited, EcoR I enzyme cutting sites and Xho I enzyme cutting sites are respectively introduced at two ends of the sequence, the gene sequence of the B438L target introduced into 1 Flag and 1 Strep at the N end is inserted into a pCMV vector through the two enzyme cutting sites to construct a pCMV-ASFV-B438L recombinant plasmid, Escherichia coli DH5 alpha competent cells are transformed, and the plasmid is plated for overnight culture. And (4) selecting positive clones for sequencing on the next day, adding glycerol into positive bacteria liquid with correct sequencing for bacteria preservation, and storing at-80 ℃ for later use.

2.3 transfection of recombinant plasmids

Using SMM 293-TI Medium and 5% CO ₂ HEK-293F cells were cultured in suspension at 37 ℃. 2mg of pCMV-ASFV-p72 plasmid, 2mg of pCMV-ASFV-B602L and 2mg of pCMV-ASFV-B438L plasmid were co-transfected with 12mg of PEI transfection reagent at a 1L density of 2X 10 ⁶ cells/mL 293F cells.

2.4 expression and purification of recombinant protein ASFV-p72

After transfection of 293F cells for 48h, the transfected cells were harvested by centrifugation at 1000 Xg for 20min, and the cell pellet was resuspended in a resuspension buffer containing 20mM hepes buffer, pH7.4, 300mM NaCl and PMSF protease inhibitor, and sonicated on ice for 3 min. 160000rpm, 4 deg.C for 20min, removing impurities from the supernatant with a 0.22 μm filter, adding affinity beads against the flag tag to the supernatant, washing the bound protein twice with resuspension buffer, and eluting the target protein with elution buffer (0.1mg/mL3 × flag peptide, 20mM HEPES and 300mM NaCl, pH 7.4). The eluate was further purified by Superdex 20010/300 GL pre-packed column, eluting the hetero-protein with 20mM imidazole buffer (20mM Tris-HCl, 300mM NaCl, 20mM imidazole, pH7.5), eluting the target protein with 200mM imidazole (20mM Tris-HCl, 300mM NaCl, 200mM imidazole, pH7.5), collecting the eluate of ASFV-p72 recombinant protein peak, and sampling each eluate in an appropriate amount for SDS-PAGE detection. Concentrating the target protein with 10KD ultrafiltration concentration tube, determining protein concentration by BCA method after the purity meets the requirement, subpackaging, and storing at-20 deg.C for use.

Purifying p72 recombinant protein by nickel column, collecting eluate of target protein peak, taking appropriate amount of sample from each tube of eluate, and performing SDS-PAGE detection, wherein the detection result is shown in FIG. 1, and a band is provided around 78KD, and the band size is in accordance with the expected target band size. The protein concentration determined by BCA method was 3.2 mg/mL.

3. Identification of recombinant p72 protein

3.1 SDS-PAGE analysis of expression products

The eluted recombinant proteins were sampled for SDS-PAGE electrophoresis.

(1) Preparing 12% separation gel (15.08 mL): purified water 5.1mL, 30% acrylamide stock solution 6.0mL, Tris-HCl (1.5moL/L, pH 8.8)3.8mL, 10% SDS 150. mu.L, 1150 μ L of 0% ammonium persulfate, 5 μ L of TEMED, ddH ₂ O make up to 15 mL. Mixing the components of the separating gel, immediately adding the separating gel solution into the mounted glass plate until the distance between the separating gel solution and the top end of the glass plate is about 1.5cm, and slightly covering a water layer on the separating gel solution to seal the top so as to prevent the inhibition effect of oxygen in the air on gel polymerization. Standing at room temperature (15-25 ℃) for about 30min, and pouring out an upper-layer capped water layer after a very obvious interface appears after gel polymerization is completed, and sucking the excessive liquid as far as possible by using filter paper.

(2) Pouring 5% concentrated glue: 1.0mL of 30% acrylamide stock solution, 750. mu.L of Tris-HCl (1moL/L, pH 6.8), 60. mu.L of 10% SDS, 60. mu.L of 10% ammonium persulfate, 6. mu.L of TEMED, in ddH ₂ O make up to 6 mL. All the components of the concentrated glue are mixed evenly, the concentrated glue solution is immediately added to the top end of the glass plate, and the glass plate is carefully inserted into a comb as soon as possible to avoid generating bubbles. Standing at room temperature (15-25 ℃) for about 30min, and carefully pulling out the comb.

(3) Electrophoresis: adding Tris-glycine buffer solution into the electrophoresis tank. Taking a proper amount of recombinant protein sample, adding equal volume of 2 xSDS loading buffer solution, mixing uniformly, boiling in water bath for 5min, loading 20 mu L, performing electrophoresis at 80V until bromophenol blue enters separation gel, increasing the voltage to 120V, and continuing electrophoresis until the bromophenol blue reaches the bottom of the gel.

(4) Dyeing: after the electrophoresis was completed, the gel was washed with purified water, soaked in a Coomassie Brilliant blue staining solution (45mL of methanol: 45mL of water: 0.25g of Coomassie Brilliant blue dissolved in 10mL of glacial acetic acid) having a volume 5 times that of the gel, and stained on a destaining shaker at room temperature (15 to 25 ℃) for 2 hours.

(5) And (3) decoloring: after washing the dyed gel with pure water, placing the gel in a mixed solution of 30% methanol and 10% glacial acetic acid, shaking for decoloring, and replacing the decoloring solution for 3-4 times. After the blue background was completely removed, the gel was immersed in pure water to terminate the decolorization.

3.2 Western-blot analysis of expression products

A sample of purified recombinant p72 protein was taken for SDS-PAGE, after which the gel was removed and a polyacrylamide gel-membrane "sandwich" was prepared in the following order: filter paper-gel-PVDF membrane-filter paper. A clean glass rod was gently rolled through the gel-film "sandwich" to eliminate air bubbles between layers. The sponge pad and filter paper, PVDF membrane were equilibrated in transfer buffer for 10min in advance. Fixing the gel-film: the sandwich is transferred into an electrotransformation instrument, the gel side faces to the negative electrode, the PVDF membrane side faces to the positive electrode, a cooling device is connected, and the 200mA constant current is transferred for 1 h. And (3) taking down the PVDF film after the transfer is finished, and carrying out the following operations:

(1) Washing the membrane I: after the transfer is finished, taking down the PVDF membrane, putting the PVDF membrane into a glass plate with a proper size, and slowly shaking and washing the PVDF membrane for 3 times by using TBST (tunnel boring machine) for 5min each time;

(2) and (3) sealing: putting the PVDF membrane into a glass plate with a proper size, and sealing with 5% skimmed milk powder at 37 ℃ for 1 h;

(3) and (3) washing a membrane II: discarding the confining liquid, and washing the membrane with TBST for 5min each time for 3 times;

(4) adding a primary antibody: adding anti-His tag monoclonal antibody (mouse source: working concentration 1:2000) diluted by confining liquid, and slowly shaking for 1h at room temperature;

(5) washing a membrane III: discarding the primary antibody, and slowly shaking and washing the membrane with TBST for 3 times, 5min each time;

(6) adding a secondary antibody: adding horseradish peroxidase-labeled goat anti-mouse IgG (diluted by 1:10000 times of working concentration) diluted by a confining liquid, and slowly shaking for 1h at room temperature;

(7) and (4) washing a membrane IV: discarding the secondary antibody, and slowly shaking and washing the membrane with TBST for 5min each time for 3 times;

(8) color development: and (3) putting the PVDF membrane into a chemiluminescence substrate solution, keeping out of the sun for color development, and imaging and photographing on a gel imager.

Western Blot identification is carried out on the purified p72 protein by using an anti-His tag monoclonal antibody, a target band appears around 75KD, and the result is shown in figure 2.

3.3 recombinant protein specificity identification

The specificity of the recombinant p72 protein is identified by using a Western Blot method. The primary antibody is respectively standard positive serum, porcine reproductive and respiratory syndrome virus (HuN4-F122 strain) positive serum, porcine circovirus type 2 inactivated vaccine (LG strain) positive serum, classical swine fever virus (C strain) positive serum, porcine pseudorabies virus (Kartha-K61 strain) positive serum, porcine negative serum and secondary antibody is goat anti-porcine IgG marked by HRP. The specific operation steps refer to 3.1 and 3.2.

The specificity of the purified recombinant p72 protein was identified by Western Blot, and the results are shown in FIG. 3. As can be seen from the figure, the p72 protein only has obvious positive reaction with standard positive serum, but has no cross reaction with porcine reproductive and respiratory syndrome virus (HuN4-F122 strain) positive serum, porcine parvovirus (WH-1) positive serum, porcine circovirus type 2 inactivated vaccine (LG strain) positive serum, classical swine fever virus (C strain) positive serum, porcine pseudorabies virus (Kartha-K61 strain) positive serum, porcine parvovirus (WH-1) positive serum and porcine negative serum, thereby proving that the recombinant protein has good specificity.

3.4 immunogenicity of recombinant proteins

Uniformly mixing 100 mu g of recombinant p72 protein with equivalent volume of Freund complete adjuvant, immunizing 2 BALB/c mice at 8 weeks old by adopting an intraperitoneal injection mode, and simultaneously emulsifying the Freund complete adjuvant and PBS to obtain 2 immune negative mice; after 14 days, carrying out second immunization, and immunizing positive mice and negative mice after emulsification of Freund incomplete adjuvant and 100 mu g p72 protein in equal volumes; after a third immunization on 28 days, 100. mu.g of recombinant p72 protein was used to immunize positive mice, and the negative mice were immunized with PBS. And respectively collecting mouse serum after the first immunization and the second immunization, collecting the mouse blood after 14 days of the third immunization, and collecting the serum.

Diluting the purified recombinant p72 protein to 1mg/mL, coating an enzyme label plate with 100 mu L per well, and coating overnight at 4 ℃; washing with PBS for 3 times, each time for 2-3 min, and blocking with 5% BSA at 37 deg.C for 2 h; washing with PBS for 3 times, each time for 2-3 min; diluting antiserum obtained after the primary, secondary and tertiary immunization of p72 with 5% BSA at a ratio of 1:100, adding the diluted antiserum into corresponding protein wells, incubating for 1h at 37 ℃, and treating the negative serum in the same way; PBST is washed for 5 times, and each time lasts for 2-3 min; adding HRP-labeled goat anti-mouse secondary antibody (dilution ratio 1:5000) diluted with 5% BSA, and incubating at 37 ℃ for 1 h; PBST is washed for 5 times, each time is 2-3 min, 100 mu L of TMB color development liquid is added into each hole, the color is developed for 15min in a dark place, and 2mol/L H is added ₂ SO ₄ The reaction was stopped and OD detected ₄₅₀ The value is obtained.

Adopt the roomAnd measuring the specific antibody titer in serum of the immunized mice after primary immunization, secondary immunization and tertiary immunization by ELISA. As shown in FIG. 4, the recombinant p72 protein induced the production of specific antibodies after immunizing mice, and the titer of the serum of the immunized mice increased with time, and the OD450 of the serum after the three-immunization was compared with the control group _nm The value can reach about 1.5, while the OD450 of the control group _nm Below 0.1.

EXAMPLE 2 preparation of murine monoclonal antibody against African swine fever Virus p72 protein

1. Material

1.1 Virus: porcine Pseudorabies live vaccine (Kartha-K61 strain) (PRV) purchased from Qingdao Yibang bioengineering Co., Ltd; porcine parvovirus inactivated vaccine (WH-1) (PPV), purchased from Pre-biological GmbH, Wuhan's family; porcine circovirus type 2 inactivated vaccine (LG strain) (PCV 2) is purchased from Harbin Vitaceae biotech development company, Swine Fever live vaccine (C strain) (Classical Swine Fever, CSF), and highly pathogenic Porcine reproductive and respiratory syndrome live vaccine (HuN4-F122 strain) (Porcine reproductive and respiratory syndrome, PRRS) is purchased from Harbin group biological vaccine GmbH, and the viruses are separated, identified and preserved from the vaccines by the animal inspection and quarantine research institute of Chinese inspection and quarantine science.

1.2 cells: SF9 cell, 293F cell, Hi5 cell and SP2/0 cell are identified, stored and supplied by animal inspection and quarantine research institute of Chinese inspection and quarantine science institute.

1.3 protein: the purified recombinant p72 protein is identified, stored and supplied by the animal inspection and quarantine research institute of Chinese inspection and quarantine science research institute; commercial His-tag proteins were purchased from Boopanke Biotech, Inc., Shanghai.

1.4 test animals: 6-8 week-old female BALB/c mice, provided by Beijing Wittingle laboratory animal technology, Inc.

1.5 related reagents: HRP-labeled goat anti-mouse IgG antibody, FITC-labeled goat anti-mouse IgG antibody, HAT medium, HT medium, and PEG1450 were purchased from Sigma; the adjuvant special for ascites and Quick Antibody-Mouse 5W adjuvant are purchased from Beijing Boolong immuno-technology GmbH; RPMI-1640 medium was purchased from GIBCO; immunoglobulin subclass identification Kit (Rapid ELISA Mouse mAb Isotyping Kit) was purchased from Thermo Scientific.

1.6. Immunizing animals: the recombinant p72 protein was diluted to a final concentration of 1mg/ml with PBS, the adjuvant was mixed well, 25. mu.L of adjuvant was taken under sterile conditions and mixed rapidly with equal volume of antigen. 10 mice were divided into two groups, and were injected subcutaneously at the back or intramuscularly at the legs. And immunizing once every two weeks and twice again, immunizing at the same dose and in the same immune mode, collecting blood 7-10 days after each immunization, and detecting the titer of the antibody generated by the mouse by using an indirect ELISA method. Selecting mice with high antibody titer, performing boosting immunization on the 3 rd day before fusion, i.e. injecting 100 mu g of antigen without adjuvant into the abdominal cavity, and taking the splenocytes of the mice to fuse with SP20 tumor cells after 3 days of impact immunization.

2. Establishment of hybridoma cell strain

2.1 preparation of SP2/0 cells

Cells were treated 3 days before cell fusion, cell supernatant was discarded, cells were resuspended in RPMI-1640 medium (containing 10% fetal bovine serum), and the cells were resuspended at 2X 10 ⁵ The amount of each cell per min was inoculated into 3-4 dishes. The SP2/0 cell status was observed 1 day before cell fusion, and the cells were perfectly round, uniform in size and smooth in edges. Before cell fusion, the cells were washed 2 times with RPMI-1640 medium and counted for use, and the number of SP2/0 cells was recorded as B.

2.2 preparation of immune splenocytes

Normal BALB/c mice were collected and blood was collected from the eyeballs (serum was collected as negative serum control). Collecting blood from eyeball of 3 days after immunization boost, soaking and sterilizing with 75% alcohol for 10min, transferring into super clean bench, cutting a small opening on mouse abdomen with surgical scissors under aseptic condition, cutting skin, exposing abdominal cavity, separating splenocytes of immunized mouse, transferring into a flat dish containing RPMI-1640 culture medium, placing on 200 mesh sieve, grinding with 5mL injector core, washing cells on two sides of the sieve with RPMI-1640 culture medium, collecting cells, transferring into 50mL centrifugal dish, collecting cells, and transferring In tubes, centrifuge at 1000rpm for 5min, repeat 1 time, wash 2 times with 1640 altogether. Counting by using a counting plate. The total number of spleen cells is generally 8X 10 ⁷ -1.2×10 ⁸ Between each other. The number of immune splenocytes was recorded as a.

2.3 cell fusion

Collecting cultured sp2/0 myeloma cells and spleen cells, B: the number of A is 1: 4-1: 10. And when the spleen cells are washed for the third time, mixing sp2/0 myeloma cells with immune spleen cells, and carrying out centrifugal cleaning together at the rotating speed of 1000rpm for 4 min.

After washing, the supernatant was decanted, a little solution was left (to ensure that the cells did not dry), and then the bottom of the centrifuge tube was flicked on the palm to loosen and homogenize the precipitated cells. Sucking 0.8mL of 50% PEG1450 pre-warmed to 37 deg.C with a 1mL pipette, holding a 50mL centrifuge tube (inclined centrifuge tube) containing mixed cells, slowly adding PEG to the mixed cells while gently rotating, adding within 1min, standing at room temperature for 1min, and introducing into a container containing PEG1450 ₂ The incubator was then used to take 50mL of 1640 incubated at 37 ℃ and add 10mL of the cell suspension slowly to the fused cells with a pipette to disperse the cell pellet, after the addition of 10mL of the cell suspension, the rest was added along the tube wall, and after the addition, the lid was tightened and turned upside down several times to mix the cells. Centrifuging at 1500r/min for 5min, discarding the supernatant, and resuspending the fused cells in HAT-containing culture medium. The number of the spread plates is calculated according to the number of sp2/0, and the number of sp2/0 in each hole is guaranteed to be 4000-10000/hole. The fused cells were added dropwise to a 96-well cell plate. At 37 ℃ with 5% CO ₂ And (5) standing and culturing in an incubator.

2.4 establishment of Indirect ELISA method

The indirect ELISA uses a matrix titration method to search the optimal coating concentration of the antigen and the optimal dilution of the enzyme-labeled secondary antibody, and the operation steps are as follows:

(1) coating: diluting the purified recombinant p72 protein to 1mg/mL, then diluting the protein to 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000 and 1:20000 by using a coating solution, respectively coating the protein, performing incubation at 2-8 ℃ overnight at 100 mu L/hole;

(2) washing: taking out the enzyme label plate, discarding the coating solution, washing for 3 times by PBST, and patting to dry;

(3) and (3) sealing: adding 200 μ L/well of 5% BSA (prepared by PBST) to each well, incubating at 37 ℃ for 1h, repeating (2) washing for 3 times, and patting dry;

(4) a first antibody: adding negative serum and positive serum diluted at ratio of 1:5000 into 100 μ L/well, setting blank control, incubating at 37 deg.C for 1h, repeating (2) washing for 3 times, and patting to dry;

(5) secondary antibody: diluting enzyme-labeled secondary antibody with diluent (i.e. blocking solution) at ratio of 1:5000, 1:10000, 1:20000, and 1:40000, reacting at 37 deg.C for 1h at a concentration of 100 μ L/well, repeating (2) washing for 3 times, and patting to dry;

(6) color development: adding 100 mu L/hole TMB color development liquid into each hole, and developing for 15min at room temperature in a dark place;

(7) terminate and read: the reaction was stopped by adding 50. mu.L/well of sulfuric acid (2moL/L) and the OD450nm value was measured by a microplate reader.

The optimal antigen coating concentration of indirect ELISA is determined to be 0.1mg/mL and the optimal dilution of the enzyme-labeled secondary antibody is determined to be 1:10000 through the square matrix titration method.

3. Preliminary screening of Positive hybridoma cell lines

When the fused cells grow to the bottom of the hole of 1/3-1/2, taking cell supernatants, and detecting and screening positive hybridoma cells by an indirect ELISA method. And continuously cloning the screened positive clones for 2-3 times by a limiting dilution method until the positive rate is 100%, carrying out amplification culture on the cell strains, and freezing and storing the positive cell strains in liquid nitrogen.

The screening method comprises the following steps:

(1) coating quilt

A: preparation of positive antigen plate: diluting the recombinant p72 protein to 1 mu g/mL by using a coating solution (carbonate buffer solution, 0.05mol/L, pH value of 9.6), coating an enzyme-linked reaction plate by using 100 mu L/hole, and standing overnight at 2-8 ℃;

b: preparation of negative antigen plate: coating an enzyme-linked reaction plate with commercial His tag protein according to 100 mu L/hole, and coating overnight at 2-8 ℃;

(2) washing the plate: spin off the liquid in the wells, wash the ELISA plate with a wash solution (PBST: 0.05% Tween-20 in PBS) at 250. mu.L/well for 3 times, and beat dry;

(3) and (3) sealing: adding blocking solution (PBST solution containing 5% BSA), 200. mu.L/well, blocking at 37 ℃ for 1h, repeating (2) plate washing for 3 times, and patting dry;

(4) Sampling: taking the supernatant of the hybridoma cells for proper dilution, adding 100 mu L/hole into an ELISA plate coated by positive antigen and negative antigen, incubating for 1h at 37 ℃, spin-drying the liquid, repeating the step (2) of plate washing for 3 times, and patting to dry;

(5) enzyme-labeled antibody: adding goat anti-mouse IgG enzyme-labeled antibody (diluted 1: 10000), 100 mu L/hole, incubating at 37 ℃ for 1h, and repeating (2) washing the plate;

(6) substrate: adding a substrate solution into the mixture, wherein the mixture is 100 mu L/hole, and incubating the mixture for 15min at room temperature (15-25 ℃) in a dark place;

(7) and (4) terminating: adding stop solution (2moL/L H) ₂ SO ₄ ) 100 mu L/hole, mixing evenly by slight shaking, and reading OD by a microplate reader under the wavelength of 450nm _450nm Values (should be read within 15min after addition of stop solution) and the results recorded.

(8) And (3) judging: the P/N is more than or equal to 2.1, and the product is judged to be positive; P/N < 2.1, and the result is judged to be negative. And sequentially judging positive hybridoma cell wells.

(9) Screening of hybridoma cells: cell clones positive for recombinant p72 protein and negative for His-tag protein were selected. After 4 times of screening, 8 hybridoma cell strains secreting African swine fever recombinant p72 protein monoclonal antibody, 12F6, 8C10, 5G8, 5C6, 18G4, 22H6, 30C3 and 24E7 are finally screened, and 8 hybridoma cells are subjected to expanded culture.

4. Indirect immunofluorescence identification of monoclonal antibodies

4.1 construction of recombinant Bacmid-ASFV-p72

The method comprises the steps of synthesizing a p72 gene full-length sequence (1941bp) of an African swine fever Heilongjiang strain (MK333180.1) published in GenBank by Beijing Optimalaceae biotechnology, Inc., inserting the p72 gene full-length sequence into a pFastBac1 vector to construct a pFastBac1-ASFV-p72 recombinant plasmid, transforming a positive recombinant plasmid pFastBac1-ASFV-p72 with correct sequencing into DH10Bac competent cells, carrying out blue-white spot screening, culturing at 37 ℃ for 48h, selecting white colonies, identifying by using an M13 universal primer, carrying out expanded culture, extracting the recombinant plasmid by using a plasmid extraction kit, and naming Bacmid-ASFV-p 72.

4.2 transfection of recombinant Bacmid-ASFV-p72 into SF9 cells

Preparing well-grown SF9 cells, transfecting Bacmid-ASFV-P72 to SF9 cells, packaging recombinant baculovirus, infecting Hi5 cells with P3 virus, paving a 96-well plate according to the cell number, continuously culturing for 48h, sucking and removing culture medium supernatant, washing twice with PBS, and fixing the cells for 15min at room temperature by using 4% paraformaldehyde; after three times of PBS washing, the mixture was left at 4 ℃ for further use.

4.3 Indirect immunofluorescence identification of monoclonal antibodies

(1) Adding a primary antibody: adding 50 mu L of hybridoma cell supernatant into each well of the fixed cells, and incubating for 1h at 37 ℃;

(2) Washing: washing with PBST for 5 times, each time for 3-5 min;

(3) adding a secondary antibody: adding FITC labeled goat anti-mouse IgG (diluted 1: 100), 50 mu L/hole, incubating for 1h at 37 ℃ in the dark, and repeating the washing step (2);

(4) and (4) observing results: the test results were observed with an inverted fluorescence microscope.

It can be seen from FIG. 5 that the green fluorescence is clearly seen for the positive control, but not for the negative control.

5. Subcloning of hybridoma cells

5.1 limiting dilution screening of subclones: the selected positive hybridoma cells were gently aspirated from the culture well, counted, diluted with an appropriate amount of HAT-containing RPMI-1640 medium (containing 10% fetal bovine serum) at 5 cells/mL, the cell suspension was dropped into a 96-well cell plate at 200. mu.L/well so that about 1 cell per well, and the plate was placed at 37 ℃ with 5% CO ₂ Culturing in an incubator.

5.2 specific detection of monoclonal antibody: and (3) taking the supernatant when the cell clone overgrows the culture holes 1/3-1/2 on 9-11 days after cloning, carrying out indirect ELISA detection according to the method, screening the cell holes with good shapes and positive detection, and continuing cloning. The cells in the positive hole can be transferred to a 24-hole culture plate, when the cells in the 24-hole plate grow well, the cells can be transferred to a flat dish for expanding culture, and the cells are frozen.

EXAMPLE 3 purification, characterization and pairing assays for monoclonal antibodies

1. Preparation of hybridoma cell strain ascites

Taking 8-10 weeks old mice bearing BALB/c, injecting ascites special adjuvant into abdominal cavity for 0.5mL, after 12-15 days, carrying out amplification culture on the screened hybridoma cell strain, collecting hybridoma cells, centrifuging at 1000rpm for 10min, resuspending with a proper amount of sterile PBS, injecting 0.2mL hybridoma cells/mouse (about containing 2.5 multiplied by 10) into abdominal cavity ⁶ Individual cell), about 10-15 days later, and after the abdominal circumference of the mouse is enlarged, collecting ascites by using a sterile syringe needle. Centrifuging the collected ascites at 10000r/min for 10min, collecting the intermediate layer, filtering with a 0.45 mu m filter to obtain the corresponding monoclonal antibody (ascites), respectively taking the supernatant according to the following steps: gradient dilution of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000 and 1:512000, and determination of ascites titer by indirect ELISA method. The titer test results are shown in table 1.

TABLE 1 Indirect ELISA test results for monoclonal antibodies

2. Purification of ascites monoclonal antibodies

2.1 octanoic acid-saturated ammonium sulfate precipitation: the operation steps of purifying the antibody by caprylic acid-saturated ammonium sulfate are as follows:

(1) pretreatment of ascites: and centrifuging the collected ascites for 30min at 8000rpm at the temperature of 2-8 ℃, and removing cell residues and small granular substances.

(2) To 1 aliquot of pretreated ascites fluid was added 4 volumes of acetic acid-sodium acetate buffer (0.06moL/L, pH 5.0) and the pH was adjusted to 4.5 with 0.1moL/L NaOH.

(3) Adding the octanoic acid dropwise while stirring at room temperature (15-25 ℃), adding 33 mu L of octanoic acid into ascites before dilution, continuing stirring at 4 ℃ for 25min after dropwise addition, centrifuging at 6000rpm at 4 ℃ for 15min, and removing the precipitate.

(4) The supernatant was filtered through a 0.45 μm filter into a new centrifuge tube, and 1/L0 volume of 10 XPBS (0.01moL/L, pH 7.2) was added, and the pH was adjusted to 7.4 with 5moL/L NaOH.

(5) And standing the supernatant for 30min at an ice bath of 2-8 ℃, adding 0.277g/mL ammonium sulfate (45% saturation), stirring while adding, continuing to stir for 30min after adding, centrifuging at 6000rpm and 4 ℃ for 15min, removing the supernatant, and collecting the precipitate.

(6) The precipitate was dissolved in 1/3 ascites volume of 1 XPBS (0.01moL/L, pH 7.2) and dialyzed against 1 XPBS buffer at 2-8 ℃ for 6h to remove residual ammonium sulfate.

2.2 purification of monoclonal antibodies by Protein G (or Protein A)

The operation is carried out according to the specification of the GE company Protein G (or Protein A) purifying column, and the specific operation steps are as follows:

(1) the collection tube was prepared and 30. mu.L of a neutralization solution (1M Tris-HCl buffer, pH9.0) was added; note: the specification shows that 30. mu.L of the neutralizing solution, but if 30. mu.L of the neutralizing solution is added, the solution is alkaline, and it is recommended that the pH value be tested in advance and how much of the neutralizing solution is added. The experiment of the last time is that 20 mu L of neutralizing solution is properly added into 400 mu L of eluent;

(2) Taking out the column, inverting the resuspension medium upside down, breaking off the lower part of the column, but not throwing away (for later use), centrifuging at 70-100g for 30 s;

(3) balancing: adding 600 μ L of binding solution (20 mM phosphate buffer solution with pH 7.0), and centrifuging for 30s at 100 g;

(4) binding of antibody: mu.L of monoclonal antibody eluate was taken. Adding antibody solution of less than 600 μ L, screwing the cap, combining for more than 20min, and mixing gently. Centrifuging for 30s at 100 g;

(5) washing: adding 600 μ L of binding solution, centrifuging for 30s at 100g, repeating, adding 600 μ L of binding solution, and centrifuging for 30s at 100 g;

(6) washing the antibody: adding 400. mu.L of washing buffer (pH 2.70.1M glycine buffer), mixing by inversion, placing the column on the collection tube, centrifuging for 30s at 70g, collecting the eluate, combining the column, mixing by inversion, placing the column on the collection tube prepared in (1), and collecting the eluate. An additional 400. mu.L of wash buffer was placed on a fresh (1) prepared collection tube and centrifuged at 70g for 30 s.

(7) Antibody concentration: and (3) centrifuging the mixture by using 1 XPBS (phosphate buffer solution) for 3 times at the temperature of 2-8 ℃ and adopting a 10KD ultrafiltration concentration tube (50mL), concentrating the antibody solution to a proper volume, transferring the antibody solution into a 1.5mL centrifuge tube, centrifuging the antibody solution for 30min at 8000rpm, removing insoluble precipitates, measuring the concentration, and subpackaging for later use.

And performing SDS-PAGE electrophoresis on the purified monoclonal antibody, photographing in a gel imaging system after the electrophoresis is finished, and recording a detection result. As shown in FIG. 6, two specific bands of the heavy and light chains of the antibody, corresponding to the expected sizes, were found at approximately molecular weights of 55kDa and 25kDa, respectively.

3. Specificity identification of monoclonal antibodies

The indirect ELISA method is adopted to determine whether the screened purified monoclonal antibody has cross reactivity with porcine pseudorabies virus (Kartha-K61 strain), porcine parvovirus (WH-1), porcine circovirus type 2 (LG strain), classical swine fever virus (C strain), highly pathogenic porcine reproductive and respiratory syndrome virus (HuN4-F122 strain) and 293F cell culture supernatant. The results are shown in Table 2.

TABLE 2 detection results of monoclonal antibody specificity

Note: "+" represents positive; "-" indicates negative.

4. Determination of the relative affinity of monoclonal antibodies

The relative affinities of the purified monoclonal antibodies were determined by indirect ELISA using the optimal antigen coating concentration and optimal secondary antibody dilution concentration determined in the assay of example 1. The purified monoclonal antibody was measured for concentration, diluted to 1mg/mL, and then diluted with a blocking solution (5% BSA) to 300. mu.g/mL, 150. mu.g/mL, 75. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL, and 1.5625. mu.g/mL, respectively, and then subjected to indirect ELISA detection. And after the reaction is finished, reading the OD450nm value, drawing a graph by using the dilution of the monoclonal antibody and the corresponding OD450nm value, taking the OD450nm when the curve is flat as 100 percent, namely the combination of the antigen and the monoclonal antibody reaches a saturation state, and taking the concentration of the monoclonal antibody corresponding to 50 percent of saturation on the curve, namely the relative affinity of the monoclonal antibody, wherein the larger the affinity is, the lower the required amount of the monoclonal antibody is. The specific results are shown in Table 3.

TABLE 3 relative affinity results for monoclonal antibodies

5. Monoclonal antibody antigen binding site analysis

5.1 determination of monoclonal antibody-antigen saturation Curve

Diluting the antigen with coating solution to 0.5. mu.g/mL, 1. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 16. mu.g/mL, 32. mu.g/mL respectively, coating overnight, washing, blocking with 5% BSA, washing, adding 100. mu.L of hybridoma cell supernatant to each well, incubating at 37 ℃ for 1h, washing, adding enzyme-labeled antibody (1:10000) with determined optimal dilution, incubating for 1h, washing, developing with TMB, reading OD value data at 450nm, and analyzing the data to obtain the antigen saturation concentration of each monoclonal antibody.

5.2 stacked ELISA analysis of monoclonal antibody antigen binding sites

Coating each strain of saturated antigen obtained by the experiment, standing overnight at 4 ℃, soaking and washing for 3 times, 3min each time; adding 200 μ L of blocking solution into each well, incubating at 37 deg.C for 1h, washing for 3 times, each time for 3 min; adding corresponding hybridoma cell supernatants, making 3 multiple wells, incubating at 37 deg.C for 1h with 100 μ L each, and washing for 3 times, each for 3 min; adding the supernatant of another hybridoma respectively, incubating at 37 deg.C for 1h, washing for 3 times, each time for 3 min; adding goat anti-mouse HRP antibody (diluted 1:10000), incubating at 37 deg.C for 1h, washing for 3 times, each time for 3 min; finally, 100. mu.L of TMB developing solution was added, the reaction was terminated for 15min and read, and the OD450nm value was recorded.

The stacking factor for each McAb combination was calculated according to the following formula:

wherein A1, A2 and A (1+2) represent McAb-1 and McAb-2, respectively, and the combined OD450nm values of the two. And (5) judging a result: when two McAb bind to different epitopes, a (1+2) ═ a1+ a2, a.i. ═ 100%; when two McAb bind to the same epitope,

when a.i > 40%, considering technical errors, two McAb are considered to recognize different epitopes.

The results of calculating the superposition coefficient (table 4) of the combination of 8 monoclonal antibodies according to the superposition ELISA method show that the superposition coefficient a.i of the combination of 5C6, 18G4, 22H6 and 24E7 is less than 40%, which proves that the 4 McAb recognizes the same antigenic site, while the superposition coefficient a.i of the combination of 12F6, 8C10, 5G8 and 30C3 is more than 60%, which proves that the 4 McAb recognizes different antigenic sites.

TABLE 4 superposition coefficients of monoclonal antibody combinations

6. Paired antibody screening by double antibody sandwich method

Antibody pairing experiments were performed using a double antibody sandwich ELISA to select the best antibody pairing combination. Ascites of the selected hybridoma cells were prepared and monoclonal antibodies were purified by the method of this example, mainly including 12F6, 8C10, 5G8, 5C6, 18G4, 22H6, 30C3, and 24E7, and were labeled with HRP using HRP Conjugation Kit labeling by Abcam, and then paired to perform a double antibody sandwich ELISA pairing experiment.

6.1 HRP-labeled monoclonal antibody

The operation is carried out according to the kit instruction, 50 mu g of monoclonal antibody is diluted into 80 mu L of PBS, 9 mu L of Modifier reagent is added, the mixture is gently mixed and then kept stand for 5min at room temperature, 10 mu L of PBS solution containing 50 mu g of HRP is added, the mixture is gently mixed and then incubated overnight at room temperature in a dark place, 10 mu L of Quencher reagent is added in the next day, the mixture is gently mixed and stopped for reaction, and the diluted use can be carried out after 30min at room temperature.

6.2 monoclonal antibody pairing experiments

The labeled 12F6, 8C10, 5G8, 5C6, 18G4, 22H6, 30C3 and 24E7 were used as detection antibodies, and the unlabeled antibody was used as a capture antibody, and the pair detection was carried out by the ELISA detection method as described above. The method comprises the following steps: the capture antibody (diluted 1: 500) is used as a coating antibody to coat the plates respectively and is kept at 4 ℃ overnight; PBST plate washing for 3 times, each time for 3 min; then adding 200 mu L of 5% BSA into each hole, and blocking for 1h at 37 ℃; PBST washing 3 times, each time 3 min; recombinant p72 antigen (diluted 1: 100) is used as the antigen to be detected, PBS is used as a negative control, 100 mu L of the recombinant p72 antigen is added into each hole, the incubation is carried out for 1h at 37 ℃, and PBST is used for washing for 3 times, 3min each time; using labeled 12F6, 8C10, 5G8, 5C6, 18G4, 22H6, 30C3 and 24E7 as detection antibodies, diluting at a ratio of 1:200, adding the diluted antibodies into an ELISA plate according to 100 mu L/hole, incubating at 37 ℃ for 1H, washing PBST for 3 times, adding TMB color development solution for 3min each time, reacting at 100 mu L/hole in a dark place for 15min at room temperature, adding 50 mu L/hole stop solution to stop the reaction, and detecting the OD450nm value by using an ELISA reader. After the detection, the P (positive control OD450nm value)/N (negative control OD450nm value) values of the capture antibody and the detection antibody of the same pair are recorded, and the best paired antibody with the largest P/N value is used.

Through the pairing experiment, the P/N result is shown in Table 5, 5G8 and 12F6 can be paired, and the pairing effect is good, wherein 5G8 is used as a capture antibody, and 12F6 labeled HRP is used as a detection antibody.

TABLE 5 monoclonal antibody pairing experimental design

7. Detection of chromosome number of hybridoma cell

Preparing chromosome after establishing hybridoma cell strain, and adopting colchicine method. Subculturing the hybridoma cells before the experiment to enable the hybridoma cells to enter a logarithmic proliferation phase; adding appropriate amount of colchicine to make final concentration 0.3mg/L, culturing for 1h, blowing cell, transferring into centrifuge tube, 1000rpm for 10min, and discarding supernatant; adding 5mL of 0.075moL/L KCL solution pre-warmed to 37 ℃, suspending and uniformly mixing the precipitated cells, and carrying out water bath at 37 ℃ for 15-20 min; adding 1mL of stationary liquid (methanol and glacial acetic acid 3: 1) prepared before use into the suspension, mixing uniformly, 10min at 1000rpm, and removing supernatant; adding 5mL of stationary liquid, suspending and uniformly mixing the cells, standing at room temperature for 20-30min at 1000rpm for 10min, and removing supernatant; repeating the operation once; then 5mL of the stationary liquid is added, the cells are suspended and mixed evenly, the tube opening is sealed, and the mixture stays overnight at 4 ℃; taking out the centrifuge tube, rotating at 1000rpm for 5min, slightly sucking and removing supernatant, leaving about 0.2-0.5mL of fixing solution according to cell volume, suspending and uniformly mixing, hanging and dripping on a glass slide just taken out of ice water, fixing by flame, and naturally drying; dyeing with newly prepared 100g/L Giemsa dye liquor for 10-20min, washing off the dye liquor with water, naturally drying, observing the chromosomes under a microscope and counting.

The number of chromosomes of SP2/0 cell was 62-68, that of spleen cell was 40, and that of hybridoma cells 12F6 and 5G8 was 102-108, which is higher than that of either parent cell, as shown in FIG. 7.

8. Determination of monoclonal antibody subclasses: hybridoma cell supernatants 5G8 and 12F6 were manipulated according to Rapid ELISA Mouse mAb Isotyping Kit instructions for the identification of antibody subclasses. The specific operation steps are as follows:

8.1 reagent configuration

(1) TBS preparation: the BumH Tristan Buffered Saline reagent was added to 500mL of ultrapure water and dissolved sufficiently.

(2)1 × Wash Buffer preparation: 30mL of 30 XWash Buffer was added to 870mlL ultra pure water and dissolved well.

8.2 sample treatment

(1) Hybridoma cell supernatant treatment: the samples were diluted with TBS1: 10. (recommended dilution range is 1: 1-1: 10).

(2) Ascites treatment: the samples were diluted with TBS1: 75000. (recommended dilution range is 1: 50000-1: 80000).

(3) Antibody treatment after purification: the samples were diluted with TBS1: 75000. (recommended dilution range is 25 ng/mL-2. mu.g/mL)

(4) Subclass identification: the TMB substrate and the strips were equilibrated to room temperature, 50 μ L of diluted sample was added to each well of 8-well strips, 50 μ L of HRP-labeled goat anti-mouse IgG + IGA + IgM was added to each well of 8-well strips, mixed well with a micro-shaker, and incubated at room temperature for 1 h. The incubation was discarded and the plate washed once with 250. mu.L of wash solution per well, then patted dry and repeated three times. After 1min, blue positive wells were observed with 75 μ L TMB substrate per well. After reacting for 5-15 min, adding 75 mu L of stop solution, and changing the color of the reaction hole from blue to yellow. When the OD450nm value was measured, the absorbance in the reaction well was more than 0.2, and it was determined to be positive.

The results of determination of the immunoglobulin subclass identification kit for the 2 selected monoclonal antibodies (ascites) are shown in fig. 8, and the antibody subclasses of the 12F6 strain and the 5G8 strain are both IgG1/κ.

9. Determination of monoclonal antibody sequences

9.1 extraction of hybridoma cell RNA

When 12F6 and 5G8 hybridoma cells grew to a monolayer, the culture broth was discarded and flushed with 3mL sterile PBS and counted, and 1X 10 cells were collected ⁶ And (3) putting the cells into a 1.5mL centrifuge tube, centrifuging for 5min at the speed of 800r/min, discarding the supernatant, and extracting the RNA of the hybridoma cells by using an RNA extraction kit according to the operation steps of the instruction.

9.2 RT-PCR amplification and identification of RNA extracted from hybridoma cells

14 degenerate primers (among them, VH-F4, VH-R2, VL-F6, VL-R2) were designed for a total of 20 pairs.

TABLE 6 heavy and light chain RT-PCR amplification primers for monoclonal antibodies

Primer and method for producing the same	Sequences (5 'to 3')
		mVL-F1	ATGGAGACAGACTCCTGCTAT(SEQ ID NO:5)
mVL-F2	ATGGATTTTCAGGTGTTTTCAG(SEQ ID NO:6)
		mVL-F3	ATGRAGTCACAKACGGTCTTYRTA(SEQ ID NO:7)
mVL-F4	ATGAGGKCCCHGCTYTYCTKGGR(SEQ ID NO:8)
		mVL-F5	ATGAAGTTGCCTGTGCTGTTG(SEQ ID NO:9)
mVL-F6	ATGATGAGTCCTGCCTTCC(SEQ ID NO:10)
		mVL-R1	ACTGGATGGTGGGAGGA(SEQ ID NO:11)
VL-R2	CCCAAGCTTACTTGGGAAGATGGA(SEQ ID NO:12)
		mVH-F1	ATGGRATGSAGCTGMATSCTCTT(SEQ ID NO:13)
mVH-F2	ATGRACTTCGGGYCTKGGTTTT(SEQ ID NO:14)
		mVH-F3	ATGGCTGTCTTGGGGCTCTTCT(SEQ ID NO:15)
mVH-F4	ATGGRCAGTACHTYY(SEQ ID NO:16)
		mVH-R1	AYCTCCACACRCCAGTGGATAGAC(SEQ ID NO:17)
VH-R2	CCCAAGCTTRCCARKGGATRA(SEQ ID NO:18)

Note: r is A/G, S is C/G, Y is C/T, M is C/A, K is T/G, H is A/T, D is G/T/A

Using the extracted RNA as a template, primers are shown in Table 6, and the reaction system is 50. mu.L (10 Xone Step RNA PCR Buffer 5. mu.L, MgCl) ₂ 10 μ L (25mM), 10 μ L of dNTP mix (10mM), 1 μ L of RNase Inhibitor (40U/μ L), 1 μ L of AMV RTase XL (5U/μ L), 1 μ L of AMV-Optimized Taq (5U/μ L), 1.5 μ L of F-equivalent mixed primer, 1.5 μ L of R-equivalent mixed primer, 4 μ L of template, ddH ₂ O15. mu.L), reaction procedure (reverse transcription at 50 ℃ for 30min, pre-denaturation at 94 ℃ for 2min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, amplification for 35 cycles, and final extension at 72 ℃ for 10 min). Finally, 8 microliter of product is used for observing the RT-PCR amplification result by 1 percent agarose gel electrophoresis, and proper primers are selected for carrying out large-scale amplification on VH and VL after identification (the step is repeated twice). The band of interest was recovered by 2% agarose gel electrophoresis, and then VH and VL genes of interest were recovered using a gel recovery kit.

9.3 ligation transformation and characterization of VH and VL

Recovering purified VH and VL target genes

Connecting, wherein the reaction system comprises the following steps: 1 μ L

The purified PCR product was recovered in 4. mu.L. The reaction was carried out at room temperature for 30 min.

50 mu L of each Trans 5 alpha competent cell is taken to melt on ice, the ligation product is added and mixed evenly, the mixture is ice-cooled for 30min, heat shock is carried out on the mixture in water bath at 42 ℃ for 45s, and ice bath is carried out for 2-3 min. Adding 600 μ L LB liquid culture medium, performing shake recovery culture at 37 deg.C for 60min at 220r/min, uniformly coating 100 μ L bacterial liquid on LB solid culture medium (AMP resistance), culturing in 37 deg.C incubator for 12-15h, and observing the occurrence of transformed single colony. 4-6 single colonies were picked into a 1.5mL centrifuge tube (600. mu.L of AMP-resistant LB broth), incubated at 37 ℃ on a shaker at 220r/min, and controls were set up.

After culturing at 37 ℃ for 4 hours, 2.0. mu.L of the bacterial suspension was collected from each centrifuge tube and subjected to PCR identification of the bacterial suspension, 20. mu.L (0.2. mu.L of rTaq enzyme (5U/. mu.L), 2.0. mu.L of 10 XPCR buffer, 2.0. mu.L of dNTP (2.5mM), 1.0. mu.L of M13-F (10. mu.M), 1.0. mu.L of M13-R (10. mu.M), 2.0. mu.L of bacterial suspension, ddH ₂ O9.8. mu.L), reaction procedure (pre-denaturation at 95 ℃ for 4min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, amplification for 32 cycles, and final extension at 72 ℃ for 10 min). And finally, taking 8 mu L of product, observing the PCR amplification result by using 1% agarose gel electrophoresis, and selecting positive clone bacterial liquid to send to sequencing.

3 positive clones identified by the light and heavy chain gene clones of the 5G8 monoclonal antibody are selected as sequencing samples to obtain the nucleotide sequence of the variable region of the antibody, and the comparison is carried out by using DNAStar and IgBLAST software, and the results show that: the 3 light chain variable region genes are 354bp in length and code 118 amino acids; the 3 heavy chain variable region gene sequences are 393bp long and code 131 amino acids. The heavy chain variable region has an amino acid sequence shown in SEQ ID NO:1 below:

among them, the heavy chain complementarity determining regions CDR1(SEQ ID NO:19), CDR2(SEQ ID NO:20), and CDR3(SEQ ID NO:21) are underlined in bold, respectively.

The light chain variable region has an amino acid sequence shown in SEQ ID NO:2 below:

Among them, the portions underlined in bold are the light chain complementarity determining regions CDR1(SEQ ID NO:22), CDR2(SEQ ID NO:23), and CDR3(SEQ ID NO:24), respectively.

Selecting 3 positive clones identified by the light and heavy chain gene clones of the 12F6 monoclonal antibody as sequencing samples to obtain the nucleotide sequence of the variable region of the antibody, and comparing by using DNAStar and IgBLAST software, wherein the results show that: the length of each of the 3 light chain variable region genes is 339bp, and 113 amino acids are coded; the 3 heavy chain variable region gene sequences are 375bp long and code 125 amino acids. The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:3 below:

among them, the heavy chain complementarity determining regions CDR1(SEQ ID NO:25), CDR2(SEQ ID NO:26), and CDR3(SEQ ID NO:27) are underlined in bold, respectively.

The light chain variable region has an amino acid sequence shown in SEQ ID NO:4 below:

among them, the portions underlined in bold are the light chain complementarity determining regions CDR1(SEQ ID NO:28), CDR2(SEQ ID NO:29), and CDR3(SEQ ID NO:30), respectively.

Example 4 establishment and application of double-antibody sandwich ELISA method based on African swine fever virus p72 gene

1. Material

1.1 preparation of reagents

(1) Coating liquid: 0.05mol/L, pH 9.6.6 carbonate buffer.

(2) Washing liquid: pH7.4, 0.1M PBS, 0.05% Tween-20.

(3) Sealing liquid: a5% BSA solution was prepared with the washing solution.

(4) Diluting liquid: namely the confining liquid.

(5) TMB: solution A: 0.02% H ₂ O ₂ Using 0.1M lemon with pH of 5.0Diluting with 0.2M acid solution of disodium hydrogen phosphate;

and B, liquid B: 0.4 per mill TMB-HCl is dissolved in 50mM sodium citrate solution with pH of 2.8.

Mixing the 50 mu L A solution and the B solution respectively, and storing in dark for later use. (commercially available TMB color developing solution may also be used)

(6) Stopping liquid: 2M H ₂ SO ₄ And (3) solution.

1.2 samples

150 swine serum samples collected between 2009-2016 were stored by the African swine fever national reference laboratory, the Poland national veterinary institute. 100 ASFV negative sera were preserved by the Chinese institute for quarantine science.

2. Establishment of African swine fever double-antibody sandwich ELISA antigen detection kit

2.1 determination of optimal Capture antibody coating concentration and enzyme-labeled antibody dilution

According to the method that the capture antibody determined in the embodiment 3 is 5G8, the enzyme-labeled antibody is 12F6(HRP-12F6), a double-antibody sandwich ELISA method is established, the recombinant P72 protein is detected, meanwhile, a negative control is set, the coating concentration of the capture antibody and the dilution of the enzyme-labeled antibody are determined by adopting a chessboard titration method, the P/N value result is calculated after the detection is finished and shown in a table 7, the highest P/N value is selected, the optimal coating concentration of the capture antibody 5G8 is determined to be 1 mu G/mL, each well is 100 mu L, and the optimal dilution of the detection antibody HRP-12F6 is 1: 500.

TABLE 7 determination of optimal capture antibody coating concentration and enzyme-labeled antibody dilution

2.2 determination of optimal blocking conditions

According to the determined optimal conditions, 1% BSA, 5% horse serum and 5% skim milk are respectively used as blocking solutions, time gradients of 30min, 60min, 90min and 120min are set for each blocking solution, 3 repetitions are set for each group, double-antibody sandwich ELISA detection is carried out, the OD450nm value is read, and the P/N value is calculated. The results are shown in Table 8, and the best blocking solution was 5% BSA with the highest P/N value and the best blocking time was 60 min.

TABLE 8 determination of optimal blocking fluid and blocking time

2.3 determination of incubation time of enzyme-labeled antibody

According to the determined optimal conditions, time gradients of 30min, 60min, 90min and 120min are set for the incubation time of the enzyme-labeled antibody HRP-12F6, 3 times are respectively carried out for each group, double-antibody sandwich ELISA detection is carried out, the OD450nm value is read, the P/N value is calculated, the result is shown in table 9, the P/N value is selected to be the highest, and the optimal incubation time of the enzyme-labeled antibody HRP-12F6 is determined to be 60 min.

TABLE 9 determination of the optimal incubation time for enzyme-labeled antibodies

2.4 determination of the development time

According to the determined optimal conditions, establishing gradients of 5min, 10min, 15min and 20min for the substrate color development time, repeating each group for 3 times, performing double antibody sandwich ELISA detection, reading OD450nm value, calculating P/N value, and finding out the result shown in Table 10, selecting the one with the highest P/N value, and determining the optimal substrate color development time to be 15 min.

TABLE 10 determination of optimal color development time

2.5 determination of the decision criterion

According to the determined optimal conditions, 100 pig serum samples which are determined to be ASFV negative are detected, and the average OD450nm is calculatedMean value

And Standard Deviation (SD), according to the formula

A positive determination criterion can be determined.

According to the detection result, cut-off value of 0.131+3 × 0.028 of 0.214 is calculated, that is, when OD450nm is more than 0.214, the sample is judged to be ASFV positive, and when OD450nm is less than 0.214, the sample is judged to be ASFV negative.

2.6 drawing of Standard Curve for double antibody Sandwich ELISA detection

Diluting a recombinant p72 protein sample from 200ng/mL2 times to 100ng/mL, 50ng/mL, 25ng/mL, 12.5 ng/mL, 6.25ng/mL, 3.125ng/mL, 1.5625ng/mL and 0.78ng/mL, detecting OD values of p72 protein standards with different concentrations by adopting the optimized optimal double-antibody sandwich ELISA program, repeating the detection for 6 times, calculating the average value and standard deviation of each concentration, and performing a difference significance analysis (t test) (the result is shown in Table 11), thereby determining that the detection range of the method is 1.5625 ng/mL-200 ng/mL; and establishing a standard curve by taking the OD values corresponding to the p72 protein standard samples with different concentrations as abscissa and taking the different concentrations of the standard samples as ordinate, and carrying out linear regression analysis by using a LINEST function in Excel software to derive a regression equation (see figure 9).

TABLE 11 OD values of different concentrations of p72 protein standards

2.7 repeatability and stability test

The recombinant p72 protein standard substance diluted by multiple times is used as a detection sample, the antibody coating plate prepared in the same batch is used, the enzyme-labeled antibody prepared in the same batch is used as a secondary antibody, 3 repeats are set for each protein dilution, double-antibody sandwich ELISA detection is carried out, and the variation coefficient of the in-batch repeatability test of the method is calculated. And (3) carrying out double-antibody sandwich ELISA detection on the diluted protein standard substance by using antibody coated plates prepared in different batches and enzyme-labeled antibodies prepared in different batches as secondary antibodies, and calculating the batch-to-batch repeatability test variation coefficient of the method. The results show (see table 12) that the intra-batch coefficient of variation is 0.46% to 5.04%, the inter-batch coefficient of variation is 0.29% to 8.43%, and both the intra-batch and inter-batch coefficients of variation are less than 10%. The method is shown to have good repeatability and high stability.

TABLE 12 double antibody Sandwich ELISA repeatability test results

2.8 specificity test

The established double-antibody sandwich ELISA is adopted to detect porcine pseudorabies virus (Kartha-K61 strain), porcine parvovirus (WH-1), porcine circovirus type 2 (LG strain), classical swine fever virus (C strain), highly pathogenic porcine reproductive and respiratory syndrome virus (HuN4-F122 strain) and recombinant p72 protein, and meanwhile, a negative control is set for specific detection. The results show (table 13), that there was no cross reaction for the remaining viruses, indicating that the double antibody sandwich ELISA method has better specificity.

TABLE 13 detection results of double antibody sandwich ELISA specificity

2.9 sample testing

The established double antibody sandwich ELISA was used to detect 150 pig sera kept by the african swine fever national reference laboratory of the polish national veterinary institute, the results of which are shown in table 14. Meanwhile, the ELISA detection result is compared with two fluorescent PCR kits, namely qPCR-Duplex quantitative (IDASF-100) of IDVet company and Virotype ASFV PCR Kit (281905) of QIAgen company, and the result shows that the coincidence rate of the Kit and the two kits can reach 98.7%.

TABLE 14 sample detection results of double antibody sandwich ELISA and fluorescent PCR

Sequence listing

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cgagattggc acaagttcgg acatgttgtt aacgccatta tgcagcccac tcaccacgca 1440

gagataagct ttcaggatag agatacagct cttccagacg catgttcatc tatatctgat 1500

attagccccg ttacgtatcc gatcacatta cctattatta aaaacatttc cgtaactgct 1560

catggtggtg gtggtggtgg ttcccccatt gaatatatgt ttataggatt aaaacctacc 1620

tggaacatct ccgatcaaaa tcctcatcaa caccgagatt ggcacaagtt cggacatgtt 1680

gttaacgcca ttatgcagcc cactcaccac gcagagataa gctttcagga tagagataca 1740

gctcttccag acgcatgttc atctatatct gatattagcc ccgttacgta tccgatcaca 1800

ttacctatta ttaaaaacat ttccgtaact gctcatggtg gtggtggtgg tggttccccc 1860

attgaatata tgtttatagg attaaaacct acctggaaca tctccgatca aaatcctcat 1920

caacaccgag attggcacaa gttcggacat gttgttaacg ccattatgca gcccactcac 1980

cacgcagaga taagctttca ggatagagat acagctcttc cagacgcatg ttcatctata 2040

tctgatatta gccccgttac gtatccgatc acattaccta ttattaaaaa catttccgta 2100

actgctcat 2109

Claims

1. An antibody that specifically binds to African swine fever virus p72 protein, wherein the antibody has heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 19, heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 20, heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 21, and light chain complementarity determining region CDR1 shown in SEQ ID NO. 22, light chain complementarity determining region CDR2 shown in SEQ ID NO. 23, light chain complementarity determining region CDR3 shown in SEQ ID NO. 24.

2. The antibody according to claim 1, wherein the antibody comprises at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 1, or a conservative variant obtained by one or more amino acid additions, deletions, substitutions or modifications to the amino acid sequence shown in SEQ ID NO. 1; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 2, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

3. The antibody according to claim 1, wherein the antibody is a monoclonal antibody and/or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a chimeric monoclonal antibody and a modified monoclonal antibody.

4. The antibody of claim 3, wherein said antibody is a murine monoclonal antibody.

5. An antibody that specifically binds to African swine fever virus p72 protein, wherein the antibody has heavy chain complementarity determining region CDR1 shown in SEQ ID NO. 25, heavy chain complementarity determining region CDR2 shown in SEQ ID NO. 26, heavy chain complementarity determining region CDR3 shown in SEQ ID NO. 27, and light chain complementarity determining region CDR1 shown in SEQ ID NO. 28, light chain complementarity determining region CDR2 shown in SEQ ID NO. 29, light chain complementarity determining region CDR3 shown in SEQ ID NO. 30.

6. The antibody of claim 5, wherein the antibody comprises at least one heavy chain variable region and at least one light chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 3, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3; the light chain variable region has an amino acid sequence shown in SEQ ID NO. 4, or a conservative variant obtained by adding, deleting, replacing or modifying one or more amino acids in the amino acid sequence shown in SEQ ID NO. 4.

7. The antibody according to claim 5, wherein the antibody is a monoclonal antibody and/or a genetically engineered antibody; the genetic engineering antibody is selected from one of a single-chain antibody, a chimeric monoclonal antibody and a modified monoclonal antibody.

8. The antibody of claim 7, wherein said antibody is a murine monoclonal antibody.

9. An African swine fever virus p72 gene double-antibody sandwich ELISA antigen detection kit, characterized in that the kit comprises the antibody of any one of claims 1-4 and the antibody of any one of claims 5-8.

10. The kit of claim 9, further comprising an elisa plate coated with monoclonal antibodies, positive and negative controls, HRP-labeled detection antibodies, sample diluent, developing solution, and washing solution.

11. Use of an antibody according to any one of claims 1 to 8 in the manufacture of a kit for detecting African swine fever virus in a sample.

12. Use according to claim 11, wherein the sample is a serum sample or a blood sample.