CN110642926B

CN110642926B - African swine fever virus p72 recombinant protein, monoclonal antibody and test paper

Info

Publication number: CN110642926B
Application number: CN201911213422.6A
Authority: CN
Inventors: 杨春江; 杨先富; 赵荣茂; 袁志波; 杨晓霞; 吴佳兴; 马孝斌; 朱琳; 于在江
Original assignee: Beijing Nabai Bio Tech Co ltd
Current assignee: BEIJING NABAI BIO-TECH Co.,Ltd.; Qiandongnan animal disease prevention and control center
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2020-04-17
Anticipated expiration: 2039-12-02
Also published as: CN110642926A

Abstract

The invention provides a recombinant protein of African swine fever virus p72, a monoclonal antibody and test paper. The amino acid sequence of the African swine fever virus p72 recombinant protein consists of SEQ ID NO: 1. SEQ ID NO: 2 is obtained by connecting flexible amino acid fragments, and has strong immunogenicity and specificity; also provided are nucleotide sequences encoding the recombinant proteins; also provides a monoclonal antibody of the African swine fever virus p72 recombinant protein obtained by immunizing animals with the recombinant protein; also provides hybridoma cells 7A7 and 3E5 with the preservation numbers of CGMCC No.18540 and CGMCC No.18539 respectively, and has high sensitivity and strong specificity to African swine fever virus p 72; also provides latex microsphere detection test paper and colloidal gold detection test paper comprising the recombinant protein antibody, and has the advantages of small batch difference, high detection sensitivity and simple operation.

Description

African swine fever virus p72 recombinant protein, monoclonal antibody and test paper

Technical Field

The invention belongs to the technical field of animal epidemic disease detection, and particularly relates to a recombinant protein of African swine fever virus p72, a nucleotide sequence, a monoclonal antibody, hybridoma cells and test paper.

Background

African Swine Fever (ASF) is a hemorrhagic, high-lethality, viral infectious disease of swine caused by African Swine Fever Virus (ASFV). All day-old pigs are susceptible to infection. The latent period of ASF natural infection is long, and the natural infection lasts for 4-19 days. The average death time after infection is 2-10 days, the clinical manifestations are hyperpyrexia, loss of appetite, bleeding of skin and internal organs, etc., and the clinical symptoms are similar to swine fever and swine erysipelas virus. After a pig or a wild pig is infected with African swine fever virus, the incubation period is usually 3-15d, the death rate of virulent strain infection can reach 100%, and the world animal health Organization (OIE) considers that investigation and diagnosis are carried out in an ASF epidemic area and an ASFV infection initial area with low toxicity, a serological method is a first-push diagnosis method, and a recombinant protein obtained by purification is used as a detection source, so that the advantages are obvious.

The African swine fever virus belongs to the genus of African swine fever virus, is a double-stranded DNA virus, and is also the only arbovirus. The ASFV genome is a double-stranded DNA with the end covalently closed, the length is 170 kb-190 kb, and the ASFV genome has an end cross-linking and inversion repeat region which codes 151-167 proteins, and the mature virus particle contains about 50 structural proteins. Wherein the nucleocapsid protein composed of p72 structural protein accounts for 1/3 of all virus proteins, and the content of the nucleocapsid protein is the most of the structural proteins. The p72 exists on the surface of the virus capsid, has better immunogenicity and antigenicity, can induce organisms to generate neutralizing antibodies, is a main antigenic area for serological detection, is also a main protein for ASFV diagnostic detection, and lays a foundation for establishing a non-infectious, rapid and sensitive serological detection method. In the binding reaction of antigen and antibody, the part where the antigen participates in binding is called the epitope of the antigen, the epitope is the basis of protein antigenicity, the epitope peptide generally adopted in the prior art is still weak in immunogenicity and poor in specificity, so that an African swine fever virus p72 recombinant antigen protein with strong immunogenicity and good specificity is needed, a monoclonal antibody or a polyclonal antibody bound with the recombinant antigen protein can be prepared according to the recombinant antigen protein, and the obtained antibody can be further used for detecting the African swine fever virus.

In addition, the traditional method for diagnosing and detecting African swine fever virus comprises a molecular biology method and an enzyme linked immunosorbent assay kit method, wherein the molecular biology method is represented by Polymerase Chain Reaction (PCR), whether animals are infected or not is confirmed by detecting virus molecules, the operation is complicated, reagents and equipment are expensive, and the operation is difficult for a pig farm; the enzyme-linked immunosorbent assay uses a 96-hole enzyme label plate as a carrier, detects whether an animal is infected with ASF virus by using an antigen-antibody specific reaction as a principle, and has the characteristics of high sensitivity, good specificity and the like, wherein the defects in the enzyme-linked immunosorbent assay are that the operation process is relatively complicated, the requirement on sample adding accuracy is high, experimental equipment such as an enzyme label instrument and a thermostat is needed to ensure the reaction environment, and the enzyme-linked immunosorbent assay is difficult to be carried out in basic-level farms, particularly in field detection; the above methods cannot achieve the purpose of rapid detection. At present, the most widely used test strip for detecting the African swine fever virus antigen is a colloidal gold test strip, the antigen-antibody reaction principle is also utilized to detect the African swine fever virus antigen, the operation is simple and convenient, the reaction time is short, but the defects are that the sensitivity is not high enough, false negative or false positive may exist, particularly, when the ASFV is rapidly detected, because samples are mostly whole blood, tissue samples and the like, the sample components are complex, long-time pretreatment cannot be carried out, the test strip may have the problems of interference, poor specificity and the like, and the requirement for rapidly detecting the ASFV is difficult to meet.

Disclosure of Invention

The technical problem to be solved by the invention is to provide the African swine fever virus p72 recombinant protein, a nucleotide sequence, a monoclonal antibody, a hybridoma cell and test paper, wherein the recombinant protein has strong immunogenicity and specificity, the antibody has strong specificity, and the test paper has high sensitivity.

In order to solve the problems, the invention provides an African swine fever virus p72 recombinant protein, wherein the amino acid sequence of the African swine fever virus p72 recombinant protein consists of SEQ ID NO: 1. SEQ ID NO: 2 are connected by flexible amino acid segments in sequence. The dominant epitope of the protein is analyzed by DNAstar and IEDB databases, so that the N-terminal epitope and the C-terminal epitope of the protein are more easily exposed, and more choices are provided for screening an antibody library.

The nucleotide sequence of SEQ ID NO: 1 (300 amino acids) as follows:

LNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQP。

the nucleotide sequence of SEQ ID NO: 2 (145 amino acids) as follows:

TWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGS。

the amino acid sequence of the flexible amino acid fragment is not particularly limited, so long as the protein fragment can be connected, the flexible amino acid fragment can be obtained through market selling, and products of different manufacturers and models do not affect the realization of the technical effect of the invention.

Preferably, SEQ ID NO: 1. SEQ ID NO: 2 is connected by an amino acid fragment GGGGS, and the amino acid sequence of the African swine fever virus p72 recombinant protein is SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 3 (total of 450 amino acids) as follows:

LNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQPGGGGSTWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGS。

the invention also provides a nucleotide sequence for encoding the African swine fever virus p72 recombinant protein.

Preferably, the nucleotide sequence is as set forth in SEQ ID NO: 4, and the sequence shown in SEQ ID NO: 4 has the following sequence structure:

ctgaatagcaggatctctaacattaaaaatgtgaacaaaagttatgggaaacccgatcccgaacccactttgagtcaaatcgaagaaacacatttggtgcattttaatgcgcattttaagccttatgttccagtagggtttgaatacaataaagtacgcccgcatacgggtacccccaccttgggaaacaagcttacctttggtattccccagtacggagactttttccatgatatggtgggccatcatatattgggtgcatgtcattcatcctggcaggatgctccgattcagggcacgtcccagatgggggcccatgggcagcttcaaacgtttcctcgcaacggatatgactgggacaaccaaacacccttagagggcgccgtttacacgcttgtagatccttttggaagacccattgtacccggcacaaagaatgcgtaccgaaacttggtttactactgcgaataccccggagaacgactttatgaaaacgtaagattcgatgtaaatggaaattccctagacgaatatagttcggatgtcacaacgcttgtgcgcaaattttgcatcccaggggataaaatgactggatataagcacttggttggccaggaggtatcggtggagggaaccagtggccctctcctatgcaacattcatgatttgcacaagccgcaccaaagcaaacctattcttaccgatgaaaatgatacgcagcgaacgtgtagccataccaacccgaaatttctttcacagcattttcccgagaactctcacaatatccaaacagcaggtaaacaagatattactcctatcacggacgcaacgtatctggacataagacgtaatgttcattacagctgtaatggacctcaaacccctaaatactatcagcccggaggaggaggttccacctggaacatctccgatcaaaatcctcatcaacaccgagattggcacaagttcggacatgttgttaacgccattatgcagcccactcaccacgcagagataagctttcaggatagagatacagctcttccagacgcatgttcatctatatctgatattagccccgttacgtatccgatcacattacctattattaaaaacatttccgtaactgctcatggtatcaatcttatcgataaatttccatcaaagttctgcagctcttacatacccttccactacggaggcaatgcgattaaaacccccgatgatccgggtgcgatgatgattacctttgctttgaagccacgggaggaataccaacccagtggtcatattaacgtatccagagcaagagaattttatattagttgggacacggattacgtggggtct。

the invention also provides a recombinant protein monoclonal antibody of the African swine fever virus p72, which is secreted and generated by the hybridoma cell 7A7 with the preservation number of CGMCCNo.18540.

The invention also provides a recombinant protein monoclonal antibody of the African swine fever virus p72, which is secreted and generated by the hybridoma cell 3E5 with the preservation number of CGMCCNo.18539.

The invention also provides a hybridoma cell which is the hybridoma cell 7A7 with the preservation number of CGMCC No. 18540.

The invention also provides a hybridoma cell which is the hybridoma cell 3E5 with the preservation number of CGMCC No. 18539.

The African swine fever virus p72 recombinant protein is used for immunizing a balb/C mouse, spleen cells of the obtained mouse are hybridized with SP2/0 tumor cells, and the screened specific hybridoma which can only react with the African swine fever virus p72 recombinant protein and does not react with swine fever virus live vaccines, porcine pseudorabies live vaccines, porcine reproductive and respiratory syndrome live vaccines, porcine parvovirus culture solutions and porcine circovirus type 2 cell culture solutions is obtained.

The invention also provides test paper for detecting the African swine fever virus, which comprises the African swine fever virus p72 recombinant protein monoclonal antibody.

Preferably, in the test paper for detecting African swine fever virus, the African swine fever virus p72 recombinant protein monoclonal antibody is marked by latex microspheres. The latex microspheres are colored polystyrene latex microspheres, and can be blue latex microspheres, red latex microspheres and the like according to dyes with different colors added when the latex is synthesized. The latex microspheres as a novel marker have the advantages of high uniformity, good monodispersity, good stability and the like, and the prepared test strip or test card has the advantages of small batch difference, high detection sensitivity and the like by using the latex microspheres as the marker.

Preferably, the monoclonal antibody of the recombinant protein p72 of the African swine fever virus is labeled by colloidal gold.

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

1. the African swine fever virus p72 recombinant protein analyzes dominant epitopes of the protein through a DNAstar and an IEDB database, so that the N-terminal epitope and the C-terminal epitope of the protein are more easily exposed, more choices are provided for screening an antibody library, the two main epitope regions are expressed in series, a flexible amino acid fragment is used as a linker, the influence of steric hindrance is reduced as much as possible while more amino acids are reserved, and the obtained recombinant protein has strong immunogenicity and specificity;

2. the hybridoma 7A7 with the preservation number of CGMCC No.18540 and the hybridoma 3E5 with the preservation number of CGMCC No.18539 are prepared by a hybridoma technology after an animal is immunized by the recombinant protein of the African swine fever virus p72 and are obtained by screening, compared with other hybridoma cells, the monoclonal antibody generated by secretion of the hybridoma has high specificity and sensitivity to the African swine fever virus p72 protein, and the African swine fever virus detection test paper with high sensitivity and strong specificity can be prepared by using the monoclonal antibody.

Biological preservation information description

The hybridoma 7A7 was deposited in the general microbiological center of China Committee for culture Collection of microorganisms 24.10.2019, having the address of No.3 Siro-1, Beijing, Toyokuo, sunny region, the microbial research institute of Chinese academy of sciences, zip code 100101, and the deposition number CGMCC No. 18540.

The hybridoma cell 3E5 was deposited in the general microbiological center of China Committee for culture Collection of microorganisms 24.10.2019, having the address of No.3 Siro-1, Beijing, Toyokuo, sunny region, the microbial research institute of Chinese academy of sciences, the postal code is 100101, and the deposition number is CGMCC No. 18539.

Drawings

FIG. 1 is a schematic structural diagram of a test paper for detecting African swine fever virus latex microspheres according to an embodiment of the present invention;

FIG. 2 is a cross-sectional view of a test strip for detecting African swine fever virus latex microspheres according to an embodiment of the present invention;

FIG. 3 is a top view of a test strip for detecting African swine fever virus latex microspheres according to an embodiment of the present invention;

FIG. 4 is an antigen identification diagram of the recombinant protein p72 of African swine fever virus obtained in the example of the invention;

FIG. 5 is a diagram showing the identification result of the cross reaction between the recombinant protein monoclonal antibody of African swine fever virus p72 and the common porcine virus obtained in the example of the present invention;

FIG. 6 is a standard curve of the African swine fever virus latex microsphere test paper obtained in the embodiment of the present invention for the quantification of the p72 protein concentration.

Wherein: 1-latex microsphere test paper for African swine fever virus; 2-a back plate; 3-nitrocellulose membrane; 4-quality control line; 5-detection line; 6-first sample pad; 7-latex microsphere pad; 8-a blood filtration membrane; 9-a second sample pad; 10-third sample pad; 11-absorbent pad.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of monoclonal antibody against recombinant protein p72 of African swine fever virus

1. African swine fever virus p72 recombinant protein gene cloning and recombinant expression vector construction

According to the Genbank published African swine fever virus p72 gene sequence (sequence number: AAT 84439.1) as reference, an optimized gene sequence is designed, in order to enable the N/C end epitope of the antigen to be more easily exposed and show more advantages, thereby screening a more appropriate monoclonal antibody, the invention analyzes the dominant epitope region of the protein sequence through DNAstar and IEDB databases, GGS is used as a linker to carry out tandem expression on the two main epitope regions, the amino acid sequences of the two epitope regions are respectively SEQ ID No.1 and SEQ ID No.2, and the influence of steric hindrance is reduced as much as possible while more amino acids are reserved. The nucleotide sequence of the optimized African swine fever virus p72 recombinant protein gene is shown as SEQ ID No.4, and the amino acid sequence of the African swine fever virus p72 recombinant protein is shown as SEQ ID No. 3.

The nucleotide sequence of SEQ ID NO: 1 has the following sequence structure:

the nucleotide sequence of SEQ ID NO: 2 has the following sequence structure:

the nucleotide sequence of SEQ ID NO: 3 has the following sequence structure:

the nucleotide sequence of SEQ ID NO: 4 has the following sequence structure:

an amplification Primer is designed by using a Primer and DNAsar, the amplification sequence of the amplification Primer is shown as SEQ ID No.4, enzyme cutting sites BamHI and SalI are introduced into the Primer, and the Primer sequence is synthesized by Shanghai. The designed primer sequences are as follows:

an upstream primer: ccggatccctgaatagcaggatctctaac

A downstream primer: acgcgtcgacctaagaccccacgtaatccgt

Inserting the obtained recombinant p72 gene into a pUC vector (finished by Beijing Optimalaceae biotechnology, Inc.), and amplifying by a PCR method by using pUC-p72 plasmid as a template, wherein the reaction conditions comprise pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1 min, 27 cycles, extension at 72 ℃ for 5min, and termination at 4 ℃, the purified PCR product and an expression vector pFastBac B are subjected to double enzyme digestion respectively, and are subjected to agarose gel electrophoresis identification, and are then connected with T4 DNA ligase at 4 ℃ overnight, and the connection product is transformed into DH5 α competent cells, and the positive recombinant plasmid is named as pFastBac HT-p 72.

2. Induction expression of African swine fever virus p72 recombinant protein

Transposing a positive recombinant plasmid into DH10Bac competent cells, screening positive clones by using KTG antibiotics and blue-white spots, extracting baculovirus plasmids (Bacmid), transfecting SF9 insect cells, observing the pathological changes of the transfected cells and collecting recombinant baculovirus. The recombinant baculovirus was infected with SF9 insect cells at MOI 0.1, centrifuged at 5000r/min for 10min after 4d and the supernatant was collected. And then SDS-PAGE electrophoresis detection is carried out, and the result shows that a specific band appears at about 50.3KDa, namely the p72 protein is successfully expressed.

3. Purification and identification of African swine fever virus p72 recombinant protein

The purification of the target protein was carried out with reference to the Ni-NTA purification System Specification (GE Co.). The purified protein was assayed for protein content using a Bradford protein concentration assay kit (Beijing kang, century Biotechnology Co., Ltd.), and was purified by SDS-PAGE and then split-filled and stored at-80 ℃ for future use.

Western Blot for identifying the antigenicity of the recombinant protein p72 of African swine fever virus:

boiling purified protein, performing SDS-PAGE electrophoresis, and transferring onto NC membrane by wet process; sealing with 5% skimmed milk powder, adding primary antibody (ASFV positive serum provided by Chinese animal epidemic prevention control center), incubating at 37 deg.C for 2h, washing with TBST for 3 times, and washing for 10 min/time; then adding a secondary antibody (rabbit anti-pig IgG HRP, 1: 3000 dilution, sigma), incubating for 1h at 37 ℃, and continuously washing for 3 times and 10 min/time by TBST; and finally adding a diaminobenzidine DAB substrate for color development, stopping the reaction by using deionized water, photographing and storing the result, and displaying a specific band at a position of 50.3KDa, wherein the right band is the recombinant protein of the African swine fever virus p72 as shown in figure 4.

The antigenicity of the recombinant protein p72 of the African swine fever virus is identified by an ELISA method:

diluting the purified African swine fever virus p72 recombinant protein with 0.05M carbonate buffer solution (pH 9.6) to 2 μ g/mL, coating an ELISA plate with 50 μ L of the protein per well, coating overnight at 4 ℃, washing the plate with PBST for 3 times, 250 μ L/well for 60s each time, sealing the ELISA plate with 200 μ L of 1% gelatin (1 g gelatin dissolved in 100mL PBS, pH 7.2-7.4, filter sterilization), and sealing for 2 hours at 37 ℃; the PBST washing plate is continued for 1 time;

adding 50 mu L of African swine fever ASFV positive serum, Classical Swine Fever Virus (CSFV) positive serum, porcine pseudorabies virus (PRV) positive serum, highly pathogenic Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) positive serum, Porcine Parvovirus (PPV) positive serum, porcine circovirus type 2 (PCV-2) positive serum, SPF porcine serum and blank PBS control into different wells (all diluted 1: 20), and incubating at 37 ℃ for 30 min; discarding the liquid in the hole, and washing the plate for 3 times;

adding 50 mu L of HRP enzyme-labeled secondary antibody into each hole, and continuously incubating at 37 ℃ for 30 min; discarding the liquid in the hole, and washing the plate for 3 times; adding TMB substrate, incubating at 37 deg.C for 10min with 50 μ L of each well, and terminating the reaction with 2M sulfuric acid; and reading the absorbance value of 450nm by using a microplate reader.

The results show the OD of the blank control and the negative control_450nmThe values should all be less than 0.25, and the OD of the positive serum_450nmThe value is more than 1.0, namely the purified recombinant p72 protein has good antigenicity and can react with ASFV positive serum, but not with CSFV positive serum, PRV positive serum, PRRSV positive serum, PPV positive serum, PCV-2 positive serum and ASFV negative serum.

4. Preparation of African swine fever virus p72 recombinant protein monoclonal antibody

The African swine fever virus p72 recombinant protein is used for immunizing a balb/C mouse according to the dose of 20 mu g/time/mouse, mixed and emulsified with equivalent dose of Freund's complete adjuvant during the first immunization, and mixed and emulsified with equivalent dose of Freund's incomplete adjuvant during the subsequent immunization. Collecting serum after multiple immunizations to measure titer, wherein the measurement result is shown in table 1, selecting 3# mice with higher serum titer, performing intraperitoneal injection for boosting immunization by using 40 mu g/mouse, removing spleen cells, fusing with SP2/0 tumor cells, and screening positive clones by using a p72 coated enzyme label plate according to a conventional method.

TABLE 1 serum titer assay results for different immunized mice

The titer of cell culture supernatant is measured after the cell culture supernatant is subjected to limited dilution to a monoclonal state, virus detection is carried out by using viruses such as a classical swine fever virus live vaccine, a porcine pseudorabies live vaccine, a porcine reproductive and respiratory syndrome live vaccine, a porcine parvovirus culture solution, a porcine circovirus type 2 cell culture solution and the like, antibody cell strains which can react with the p72 recombinant protein of the nonhorizonum classical swine fever virus and do not react with other viruses are screened to obtain a plurality of cell strains such as 7A7, 3E5, 2H3, 3E1, 5A3 and the like, after the screened hybridoma cell strains are subjected to amplification culture, 0.2mL (containing 2.5 multiplied by 106 cells) of female BALB/c mice pretreated by a Freund's incomplete adjuvant are injected into abdominal cavities, and after about 10 to 15 days, ascites is collected by using a sterile syringe needle when the abdominal cavity of the mice is obviously swollen. The collected ascites was centrifuged at 1000 r/min for 10min, and the intermediate layer was collected.

Ascites fluid was centrifuged at 12000 r/min for 5min, the supernatant was taken and diluted with binding buffer at a ratio of 1: 7. The diluted sample was filtered using a filter and then applied to the column (Protein G was previously equilibrated with binding buffer). After the column loading was complete, approximately 5 column volumes were washed with binding buffer; then eluted with elution buffer (0.1M glycine adjusted pH 2.7 with hydrochloric acid) and the eluate at A280 was collected. Glycine was then rapidly neutralized with 1M Tris-HCl (pH 9.0) solution to bring the pH to the neutral range. The purified antibody was dialyzed into 1 × PBS, during which multiple changes were made. Quantitatively subpackaging the monoclonal antibody qualified in the inspection, storing at below-80 ℃ in a tube of 1mL, and avoiding repeated freeze thawing and pollution.

5. African swine fever virus p72 recombinant protein monoclonal antibody immunospecific identification

Specific identification of monoclonal antibodies 7a7 and 3E 1:

the ASFV positive spleen grinding fluid, the ASFV negative spleen grinding fluid, PRV, PCV2, PRRSV, CSFV and the like are coated on an enzyme-linked reaction plate and respectively react with 2 strains of monoclonal antibodies, and the result shows that the monoclonal antibodies 7A7 and 3E1 and the ASFV positive spleen grinding fluid have specific reaction, the OD450nm value is more than 1.0, but the monoclonal antibodies do not react with the negative spleen grinding fluid and other viruses, which indicates that the monoclonal antibodies 7A7 and 3E1 have good specificity, and the detailed description is shown in FIG. 5.

6. Pairing of two monoclonal antibodies

Diluting the screened monoclonal antibody to 1 microgram/ml by using a pH 9.6 carbonate buffer solution, coating overnight at 4 ℃, washing the plate for 3 times by using PBST, then blocking for two hours at 37 ℃ by using a blocking solution containing 1% BSA, adding African swine fever virus p72 recombinant protein, reacting for 30min at 37 ℃, and washing the plate for 5 times; adding intoHRP-labeled enzyme-labeled mouse monoclonal antibody (diluted by 1:10000 times), reacting at 37 ℃ for 30min, and washing the plate for 5 times; adding TMB developing solution for developing for 10min, adding stop solution, and reading OD_450nmThe results are shown in Table 2.

TABLE 2 different antibody pairing results and OD_450nmValue of

Selection of OD_450nmTwo pairs of monoclonal antibodies with a value greater than 2.0, 7A7 and 3E5, 2H3 and 3E1, respectively, were used for 10 pairs of monoclonal antibodies by the ELISA method described above^-2、10^-3、10^-4、10^-5Three positive quality control products of P72 protein of 0.1 mg/mL with different dilutions were subjected to sensitivity detection, and OD was read_450nmThe results are given in Table 3.

TABLE 32 detection results for paired antibody sensitivity

As can be seen from the results in table 3: for P72 protein positive quality control diluted by different fold ratios, 7A7 and 3E5 paired monoclonal antibodies are used for detecting OD_450nmThe values are higher than those of the paired monoclonal antibodies 2H3 and 3E1, which shows that the sensitivity of 7A7 and 3E5 is higher, the optimal paired monoclonal antibodies are determined to be 7A7 and 3E5, and the optimal paired monoclonal antibodies are used as a marker antibody and a capture antibody of the African swine fever virus antigen latex microsphere detection test strip.

Example 2 preparation of latex microsphere-labeled African Swine fever Virus p72 recombinant protein monoclonal antibody

Diluting the prepared African swine fever virus p72 recombinant protein monoclonal antibody (7A 7) with a diluent, and then labeling with latex microsphere particles, wherein the specific operations are as follows:

1. cleaning the latex microspheres: latex microspheres (purchased from Suzhou Biotechnology Co., Ltd., product No. DR 05C) having a certain volume and a particle size of 300nm were measured and poured into a clean centrifuge tube, and 900. mu.L of a labeling buffer (50 mM MES, pH 6.0) was added to 100. mu.L of the latex microspheres. Centrifuging at 17000 r/min for 10min, removing supernatant, adding 1000. mu.L labeling buffer solution, centrifuging at 17000 r/min for 10min, removing supernatant, and adding 1000. mu.L labeling buffer solution to resuspend the microspheres.

2. Activating the latex microspheres: 20 mg of NHS and EDC are respectively weighed and dissolved by 1mL of marking buffer solution (50 mM MES, pH6.0), and the solution is prepared in situ to respectively obtain 20 mg/mL of NHS solution and 20 mg/mL of EDC solution; adding 20 mu L of prepared NHS solution into the cleaned microspheres, and quickly and uniformly mixing; then 5 mul of prepared EDC solution is added into the microspheres and mixed quickly; incubating at room temperature for 20-30 min.

3. Marking the latex microspheres: centrifuging the activated latex microspheres obtained in the step 2 at 17000 r/min for 10min, and adding 1000 mu L of labeled buffer solution to resuspend the microspheres; centrifuging at 17000 r/min for 10min, discarding the supernatant, and adding 1000 μ L labeled buffer solution to resuspend the microspheres; a certain amount of monoclonal antibody (7A 7) is measured, the activated microsphere buffer solution is added according to the proportion that 100 mu L of microsphere buffer solution is added into each 0.1 mg of antibody, and after the mixture is quickly mixed uniformly, the mixture is incubated for 3 hours at room temperature.

Then measuring 100 mul volume of latex microspheres, adding blocking solution (20 mg/mL BSA, fully dissolved by 100 mM ethanolamine solution with final concentration), and incubating for 1 hour at room temperature; centrifuging at 17000 r/min for 10min, discarding the supernatant, adding 1000 μ L buffer solution to resuspend the microspheres, repeating twice, and removing unbound antibody. And finally, resuspending the microspheres with 1000 mu L of labeling buffer solution to obtain an antibody-microsphere labeling compound, and standing at 4 ℃ for later use, wherein a solution with the final concentration of 0.2% BSA and 0.02% NaN3 is added if the antibody-microsphere labeling compound is stored for a long time.

EXAMPLE 3 preparation of latex microsphere mats

1. And taking out the latex microsphere marked African swine fever virus p72 recombinant protein monoclonal antibody solution from a refrigerator at 4 ℃, and returning to room temperature.

2. A20 cm × 30cm glass fiber is taken and cut into 0.6 cm × 30cm specification by an auxiliary material strip cutting machine.

3. Spreading a layer of preservative film on a table, placing the cut glass fiber on the preservative film, and sucking 1mL of the African swine fever virus p72 recombinant protein monoclonal antibody solution marked by the latex microspheres by using a pipette to uniformly wet the cut glass fiber. And airing at room temperature for 12-16 hours, transferring to a drying oven at 40 ℃ for 1 hour, then placing in a sealing bag filled with a drying agent, and sealing for later use.

EXAMPLE 4 preparation of nitrocellulose Membrane

The capture antibody (3E 5) and the quality control line goat anti-mouse antibody were diluted to 0.5mg/ml with PBS buffer solution of pH 7.20.01M, and sprayed on a nitrocellulose membrane as a detection line 5 and a quality control line 4 using a streaking instrument.

EXAMPLE 5 preparation and examination of blood filtration Membrane

1. Preparing a blood filtering membrane: collecting blood filtering membrane, inspecting and removing blood filtering membrane with foreign matter, breakage and stain, and cutting into specification (30 cm × 12 mm) with auxiliary material slitter.

2. And (3) testing a blood filtering membrane: 1) the appearance surface is clean and tidy without impurities; no obvious difference in transparency should be seen when viewed against the illuminant; 2) cutting 1cm blood filtering membrane (0.5 cm × 2 cm) with water ductility, placing on a support, and slowly dripping whole blood sample from one end with pipette until the whole blood filtering membrane is just saturated, wherein the blood filtering amount can reach 120 + -5 μ L/cm²。

EXAMPLE 6 treatment of sample pad and absorbent pad

The latex microsphere test paper for detecting African swine fever virus is suitable for detecting whole pig blood, serum, tissue samples and the like, and does not need complex pretreatment. Because components such as whole blood, tissue samples and the like are complex, the interference on test results is easy to cause, and the specificity is poor, the invention uses 3 layers of sample pads and different buffers for processing while using the blood filtering membrane, and the optimal processing method is determined by detecting the sensitivity, the specificity and the coincidence rate as follows:

1. treatment of the first sample pad 6: a30 cm × 6 mm glass fiber sample pad is soaked in 1 mL/strip of 0.02M PB (pH 7.2) solution and dried in an oven at 40 ℃ +/-2 ℃ for 16 h for later use.

2. Treatment of the second sample pad 9: a30 cm × 15mm glass fiber sample pad is soaked in 2.5 mL/strip of 0.02MPB (pH 7.2) solution containing 2% sucrose, and is dried in an oven at 40 ℃ +/-2 ℃ for 16 h for later use.

3. Treatment of the third sample pad 10: a30 cm-23 mm glass fiber sample is soaked in a 0.02M PB (pH7.2) solution containing 2% of sucrose and 0.5% of Tween 20 in 5 mL/strip and dried in an oven at the drying temperature of 40 +/-2 ℃ for 16 hours for later use.

4. Treatment of the absorbent pad 11: the absorbent paper is cut into 30cm × 18 mm by a paper cutter for standby.

EXAMPLE 7 Assembly of the test strips

The test strip is specifically assembled as shown in fig. 1, 2 and 3, and the treated nitrocellulose membrane 3, the latex microsphere pad 7, the blood filter membrane 8, the

sample pads

6, 9 and 10, the absorbent paper 11 and other related raw materials are taken out in a clean environment with the humidity of 30% and the temperature of 18-26 ℃. The dried and fixed nitrocellulose membrane 3 is stuck on a PVC (polyvinyl chloride) back plate 2, and a sample pad, a latex microsphere pad and absorbent paper are stuck to obtain the African swine fever virus latex microsphere detection test paper 1, wherein the sticking method comprises the following steps:

1. pasting a first sample pad 6 (30 cm × 6 mm specification): the upper edge of the first sample pad 6 needs to be pressed against the lower edge of the nitrocellulose membrane 3, and the first sample pad 6 and the nitrocellulose membrane 3 are overlapped by 1.5-2.0 mm.

2. Pasting a latex microsphere cushion 7: the upper edge of the latex micro-ball pad 7 is required to press the lower edge of the first sample pad 6, and the latex micro-ball pad 7 is overlapped with the first sample pad 6 by 1-1.5 mm.

3. Pasting a blood filtering membrane 8: the blood filtering membrane 8 is pasted below the latex microsphere pad 7 and is pressed flat by hand, and the overlapping of the blood filtering membrane 8 and the latex microsphere pad 7 is required to be 2.5-3.0 mm.

4. Second sample pad 9 (30 cm x 15mm gauge) was applied: a second sample pad 9 of 30cm x 15mm is attached under the blood filtration membrane 8 and flattened by hand, requiring the second sample pad 9 to overlap the blood filtration membrane 8 by 9.0 to 9.5 mm.

5. Apply a third sample pad 10 (30 cm x 23 mm gauge): a third sample pad 10 of 30cm x 23 mm gauge is applied to the underside of the second sample pad 9 and flattened by hand, requiring the lower end of the third sample pad 10 to be aligned with and held against the lower end of the PCV floor 2.

6. Pasting the water absorption pad 11: and (3) uncovering the uppermost double-sided adhesive tape of the bottom plate 2 to which the third sample pad 10 is adhered, adhering the cut absorbent pad 11 above the membrane along the nitrocellulose membrane 3, and aligning and adhering the upper end of the absorbent paper with the upper end of the PVC bottom plate 2.

7. Slitting: trimming the assembled board, sending the board into a slitter, and cutting the board into test strips with the width of 4.0 +/-0.1 mm.

8. Card installation: the test paper strips without scratches, stains and neat edges are picked up and put into the bottom card of the card, the cover of the card shell is covered, and the test paper strips are sent into a capping machine to be capped.

9. Bagging: and putting the pressed test strip and 1 grain of drying agent into an aluminum foil bag.

10. Sealing and labeling: and (4) putting the test paper strips filled with the bags into a sealing machine, and sealing. And (5) sticking a label at the center of the sealed aluminum foil bag.

Example 8 application method of latex microsphere test paper for African swine fever virus and result determination

1. Treatment of whole blood, plasma and serum: unprocessed fresh whole blood or whole blood treated by adding an anticoagulant can be used as a detection sample, and whole blood separated plasma or serum (centrifuged at 5000-8000 r/min for 2-5 min or naturally separated overnight at 4 ℃) can be collected as the detection sample.

2. Tissue organ sample treatment: sampling from three different positions of tissues such as spleen, lymph node and the like respectively, weighing about 1g of sample, shearing by using an operation, uniformly mixing, grinding 0.1 g in a grinder, adding 1.5mL of physiological saline for continuous grinding, transferring to a 1.5mL sterilization centrifugal tube after homogenizing, centrifuging at 8000 r/min for 2 min, taking 100 mu L of supernatant to be placed in the 1.5mL sterilization centrifugal tube, and numbering for later use.

3. Tearing the test strip aluminum foil bag for packaging, taking out the test strip, and placing the test strip on a flat and clean test table.

4. The sample was aspirated with a disposable plastic pipette, 1 drop (about 30 μ L) was added to the well, followed by 3 drops of sample diluent (about 120 μ L) were slowly added vertically.

5. And after the sample diluent is added dropwise, waiting for 10min to judge the result, and invalidating the result after 20 min.

6. And (4) judging the result: when the C line of the test strip is colored and the T line is not colored, the detection result of the sample is negative. When the C line and the T line of the test strip are developed, the detection result of the sample is positive, and the deeper the T line is, the stronger the positive is. When the C line of the test strip does not develop color and the detection result of the sample is invalid, the test strip should be replaced for retesting.

Example 9 detection of sensitivity, specificity, homogeneity and stability of latex microsphere test strips for African swine fever Virus

1. And (3) sensitivity detection: adopting healthy piglet anticoagulation (African swine fever virus negative detected by an African swine fever virus fluorescence PCR detection kit (veterinary drug Sheng word 010628858)) as negative quality control; the protein concentration of the African swine fever virus p72 recombinant protein expressed in vitro is measured by an ultramicro spectrophotometer, and the anticoagulated blood of healthy piglets is diluted to 0.25 mg/mL to be used as positive quality control.

To 10^-3、10^-4、10^-53 positive quality control samples with different dilutions and 1 negative quality control sample are respectively detected by the African swine fever virus antigen latex microsphere detection test paper card. The results of the measurements are shown in Table 4 below.

TABLE 4

The results show that: test paper card pair 10 of the present invention^-5The detection result of the positive quality control sample is positive, which indicates that the detection limit of the test paper card on the recombinant protein of the African swine fever virus p72 can be 2.5ng/mL at least.

2. And (3) specific detection: the test paper card is characterized in that 5 specific quality control samples are detected by taking live vaccines of porcine reproductive and respiratory syndrome (purchased from Mediterranean stock Co., Ltd., JXA1-R strain), live vaccines of hog cholera virus (purchased from Wuhan pre-biological stock Co., Ltd., CVCC AV1412 strain), live vaccines of pseudorabies (purchased from Wuhan pre-biological stock Co., Ltd., HB-98 strain), culture solution of porcine parvovirus (purchased from Chinese veterinary microbial strain preservation center), and culture solution of porcine circovirus type 2 cells (provided by animal husbandry and veterinary research institute of Beijing academy of agriculture and forestry), and the results are negative, which indicates that the test paper card does not have cross reaction with other porcine viruses and has good specificity.

Uniformity: get 10^-5The positive quality control samples are repeatedly detected for 10 times, the results are positive, and the color development is uniform; and (3) taking the negative quality control sample for repeated detection for 10 times, wherein the results are negative and the color development is uniform.

Stability: the test paper strips are placed at 37 ℃ for 7 days, and the test of each index is respectively carried out, which meets the requirements.

Example 10 quantitative determination of latex microsphere test paper for African swine fever Virus

1. Adopting healthy piglet anticoagulation (African swine fever virus negative through African swine fever virus fluorescence PCR detection kit (veterinary drug Sheng 010628858)); the protein concentration of the African swine fever virus p72 recombinant protein expressed in vitro is measured by an ultramicro spectrophotometer, and the anticoagulated blood of healthy piglets is diluted to 0.25 mg/mL to be used as positive quality control.

2. Diluting the positive quality control substances to the concentrations of 0ng/mL, 2.5ng/mL, 50ng/mL, 100ng/mL and 300ng/mL respectively, detecting by using the African swine fever virus latex microsphere detection test paper card, reading the color development value of the detection line by using an NB reader after 10min, and drawing a standard curve, wherein the standard curve is shown in figure 6.

3. The positive quality control substances are diluted to the concentrations of 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL, the positive quality control substances are respectively detected by using the test paper card, the color development value is read by using a reading instrument, the concentration of the p72 protein in the sample is calculated according to a standard curve and is compared with the actual concentration, and the comparison result is shown in Table 5.

TABLE 5 measurement of p72 concentration in samples

As can be seen from the results in Table 5, the concentration of the African swine fever virus p72 protein in the sample obtained by the determination and calculation of the invention has no obvious difference from the actual concentration, which indicates that the test strip of the invention can be used for quantitative detection of the concentration of the African swine fever virus p72 protein in the sample, and can also be used for evaluating the content of the African swine fever virus antigen in the pig blood.

EXAMPLE 11 comparison of sensitivity of the latex microsphere test strip of the invention to that of the colloidal gold test strip

1. Preparing colloidal gold particles: reducing chloroauric acid into 20-40 nm colloidal gold particles by using a trisodium citrate reducing agent, taking 500-1000 ml of chloroauric acid aqueous solution, heating the chloroauric acid aqueous solution to boiling by using a constant-temperature electromagnetic stirrer, adding 0.5-1.5 ml of 16% trisodium citrate aqueous solution under the condition of continuous stirring, and continuously stirring and heating for 5-10 min to obtain a transparent red solution. Cooling at room temperature, restoring the original volume with deionized water, and storing at 4 ℃.

2. Preparation of African swine fever virus p72 recombinant protein monoclonal antibody-colloidal gold marker: taking 1mL of the colloidal gold prepared in the previous step, adding 2.5-4.5 mu L of K with the concentration of 0.1mol/L₂CO₃Adjusting the pH value of the solution, adding 0.8-1.5 mg of screened and purified African swine fever virus p72 recombinant protein monoclonal antibody (7A 7), uniformly mixing, standing for 5min, adding 10 mu L of 10% polyethylene glycol 20000 (PEG, Wakay, Cat. 20170615) solution, centrifuging at 12000rpm for 7min, discarding the supernatant, adding 100 mu L of complex solution (0.05M tris (hydroxymethyl) aminomethane +5% sucrose), and uniformly mixing.

3. Assembling the test strip: the procedure is as in example 6, replacing the latex microsphere pad with the gold gel pad.

4. Sensitivity contrast test for latex microsphere test paper card and colloidal gold test paper strip

To 10^-3、10^-4、10^-5In total, 3 positive quality control samples with different dilutions and 1 negative quality control sample were tested by using the African swine fever virus latex microsphere test paper and the African swine fever virus colloidal gold test paper of the invention, and the test results are shown in the following Table 6.

TABLE 6

As can be seen from the detection results: the invention relates to a test paper for detecting African swine fever virus latex microspheres and a test method thereofThe colloidal gold test paper prepared from the same raw materials is used for respectively detecting p72 protein with different concentrations, and the latex microsphere test paper card is used for detecting 10^-5The diluted p72 protein is positive, the sensitivity reaches 2.5ng/mL, and the colloidal gold test strip detects 10^-4The diluted p72 protein is positive, and the detection is 10^-5The diluted p72 protein was negative, and the sensitivity was 25ng/mL, which was significantly lower than that of the latex microsphere paper.

Example 12 comparison of the coincidence rates of latex microsphere test paper and colloidal gold test paper with a fluorescent PCR kit

720 parts of cloaca swabs and clinical samples of 12 SPF pigs attacking African swine fever virus and 12 SPF pigs of a control group are respectively detected by using an African swine fever virus fluorescent PCR detection kit (veterinary medicine, Japanese patent application No. 010628858), the self-made African swine fever virus latex microsphere detection test paper and colloidal gold test paper.

The detection results show that 129 parts of positive and 591 parts of negative are detected by the fluorescence PCR detection kit, 123 parts of positive and 597 parts of negative are detected by the African swine fever virus specific antigen detection test paper card, 115 parts of positive and 605 parts of negative are detected by the colloidal gold test paper strip, 117 parts of positive samples are detected by the latex microsphere test paper card and the fluorescence PCR detection results, 585 parts of negative samples are detected by the latex microsphere test paper card, the positive coincidence rate is 90.70%, the negative coincidence rate is 98.98%, and the total coincidence rate is 97.50%. The test results of the colloidal gold test strip and the fluorescence PCR test result are 105 parts of positive samples, 581 parts of negative samples, 81.40% of positive coincidence rate, 98.31% of negative coincidence rate and 95.28% of total coincidence rate, and the specific results are shown in tables 7 and 8.

TABLE 7 Total coincidence results of latex microsphere test paper detection and fluorescence PCR detection of the present invention

TABLE 8 Total coincidence results of colloidal gold test strip detection and fluorescent PCR detection

As can be seen from the detection results: the coincidence rate of the African swine fever virus latex microsphere detection test paper and the fluorescent PCR kit is obviously higher than that of a colloidal gold test paper prepared by the same monoclonal antibody, which shows that the African swine fever virus latex microsphere detection test paper has higher accuracy.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

SEQUENCE LISTING

<110> Beijing Nabai Biotechnology Ltd

<120> African swine fever virus p72 recombinant protein, monoclonal antibody and test paper

<130>2019

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Claims

1. The African swine fever virus p72 recombinant protein monoclonal antibody is secreted and produced by hybridoma cell 7A7 with the preservation number of CGMCCNo.18540, and the N end to the C end of an amino acid sequence of the African swine fever virus p72 recombinant protein consist of SEQ ID NO: 1. SEQ ID NO: 2 are connected by flexible amino acid segments in sequence.

2. The African swine fever virus p72 recombinant protein monoclonal antibody is secreted and produced by hybridoma cell 3E5 with the preservation number of CGMCCNo.18539, and the N end to the C end of an amino acid sequence of the African swine fever virus p72 recombinant protein consist of SEQ ID NO: 1. SEQ ID NO: 2 are connected by flexible amino acid segments in sequence.

3. A hybridoma cell, which is characterized in that the hybridoma cell is a hybridoma cell 7A7 with the preservation number of CGMCC No. 18540.

4. A hybridoma cell, which is hybridoma cell 3E5 with the preservation number of CGMCC No. 18539.

5. A test strip for detecting African swine fever virus, which comprises the African swine fever virus p72 recombinant protein monoclonal antibody of claim 1 or 2.

6. The test strip for detecting African swine fever virus according to claim 5, wherein the monoclonal antibody against the p72 recombinant protein of African swine fever virus is labeled with latex microspheres.

7. The test strip for detecting African swine fever virus according to claim 5, wherein the monoclonal antibody of the P72 recombinant protein of African swine fever virus is labeled with colloidal gold.