CN109254155B - Colloidal gold immunochromatographic test paper for detecting African swine fever virus antigen, and preparation method and application thereof - Google Patents
Colloidal gold immunochromatographic test paper for detecting African swine fever virus antigen, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a colloidal gold immunochromatographic test paper for detecting an African swine fever virus antigen, a preparation method and application thereof, belonging to the field of virus epidemic disease diagnosis technology and animal quarantine. Wherein the colloidal gold pad is internally provided with a monoclonal antibody Mab1 of one epitope of the anti-ASFV p30 antigen marked by colloidal gold; the surface of the nitrocellulose membrane is marked with a detection line and a quality control line, the detection line is a monoclonal antibody Mab2 aiming at another epitope of the ASFVp30 antigen, and the quality control line is a goat anti-mouse IgG antibody. The colloidal gold immunochromatographic test strip established by the invention has the advantages of convenience, rapidness, sensitivity, specificity and the like, can be used for directly detecting infected ASFV cells, and is suitable for ASFV infection diagnosis, epidemiological investigation and pig international trade quarantine inspection.
Description
Technical Field
The invention belongs to the field of virus epidemic disease diagnosis technology and animal quarantine, and particularly relates to a colloidal gold immunochromatographic test paper for detecting an African swine fever virus antigen, and a preparation method and application thereof.
Background
African Swine Fever (ASF) is a highly-contagious infectious disease of pigs, the death rate of the highly-pathogenic ASFV infected domestic pigs can reach 100%, the disease is popular in many African countries, is spread to adjacent countries such as Gruzia, Immunia, Ashebiejiang and Russia at present, and has a great threat to the pig industry in China. Because no vaccine is used for ASF epidemic prevention at present, rapid and accurate diagnosis is very important for preventing the spread and the epidemic of the disease. The world animal health Organization (OIE) recommended ASF diagnostic techniques include pathogen detection, nucleic acid identification, and serological testing.
In the common technology of pathogen detection, the blood adsorption test is one of the classical ASF diagnosis methods, has higher sensitivity, but needs to temporarily prepare and culture the pig bone marrow cells, not only wastes time and is fussy to operate, but also can not be used for the diagnosis of non-blood adsorption strains; the fluorescence antibody test for detecting ASFV antigen can be used for detecting suspicious pig tissue and leucocyte sample, is not only suitable for identifying non-blood-adsorption strain, but also can be used for differential diagnosis with other virus infection, but needs to prepare frozen section or smear, has great technical difficulty, and can only be carried out in special ASF diagnosis reference laboratory.
The common methods for ASFV nucleic acid identification are Polymerase Chain Reaction (PCR) and real-time quantitative PCR, which have the advantages of rapidness, sensitivity and the like, are also suitable for virus detection of putrefactive samples, have become main ASF diagnostic techniques, but require expensive instruments and anti-pollution measures, and sometimes the detection result needs to be verified by methods such as sequence determination and the like.
In the serological detection technology, ASFV infected pigs can generate specific antibodies 7-10 days after infection and can maintain for a long time, so indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and other methods can be used for detection. Among them, ELISA is an international trade inspection method specified by OIE, but detection antigens need to be prepared from virus-infected cells, virus culture causes virus diffusion risk, diagnostic reliability needs to be improved, and results need to be verified by methods such as Western-blotting (Western-blotting). In order to solve the problems, recombinant antigen ELISA is tried at home and abroad, commercial kits are available in France and Spain, but the preparation difficulty of the recombinant antigen is high, so that the kit is limited in supply and high in price, and the detection results of virus strains in different regions need to be further verified.
Disclosure of Invention
In order to overcome the defects, the invention provides the colloidal gold immunochromatographic test paper for detecting the African swine fever virus antigen, which has high accuracy and high detection efficiency, and the preparation method and the application thereof.
The applicant develops a multi-antigen indirect ELISA kit and colloidal gold test paper aiming at an ASFV antibody, and the test result shows that the kit has higher detection accuracy.
ASFV p30 is a 30kD protein encoded by ASFV ORF CP204L, and is packaged into virions at the early stage of viral infection after phosphorylation of serine residue at the amino terminal end of p30 expressed in large numbers in cytoplasm; p30 has excellent antigenicity, can induce body to produce strong humoral immune response, and stimulate animal body to produce antibody with certain neutralizing effect. According to the characteristics of early expression and large expression quantity of p30 in cell cytoplasm, the invention establishes double-antibody sandwich ELISA colloidal gold immunochromatographic test paper by using two prepared monoclonal antibodies aiming at different epitopes of p30, and detects p30 in cells, thereby determining whether animals are infected with ASFV. The colloidal gold immunochromatographic test paper can obtain a definite diagnosis result in a short time, can be used for detecting p30 antigen in porcine peripheral blood mononuclear cells and ASFV in-vitro infected cells, and is particularly suitable for ASFV infection diagnosis, epidemiological investigation and swine international trade quarantine inspection.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that:
anti-ASFV p30 monoclonal antibody Mab1, hybridoma cell secreting monoclonal antibody Mab1 was deposited with the Chinese type culture Collection with the address: wuhan university, the preservation number is: CCTCC NO, C2018158, and is named in classification as: hybridoma cell line 6G12, with a preservation date of: 2018.9.5, respectively; the epitope which the anti-ASFV p30 monoclonal antibody Mab1 is directed against is located at p30aa145-aa 154.
The anti-ASFV p30 monoclonal antibody Mab2, hybridoma secreting monoclonal antibody Mab2 is deposited in China center for type culture Collection, at the address of university of Wuhan, China, with the deposit number: CCTCC NO, C2018159, and is named in classification as: hybridoma cell line 3H6, with a preservation date of: 2018.9.5, respectively; the epitope which the anti-ASFV p30 monoclonal antibody Mab2 is directed against is located at p30aa145-aa 154.
anti-ASFV p30 monoclonal antibodies Mab1 and Mab2, and hybridoma cells secreting monoclonal antibody Mab1 were deposited with the Chinese type culture Collection with the addresses: wuhan university, the preservation number is: CCTCC NO, C2018158, and is named in classification as: hybridoma cell line 6G12, with a preservation date of: 2018.9.5, respectively; the hybridoma cells secreting monoclonal antibody Mab2 were deposited at the chinese type culture collection, at the address of wuhan university, with the following deposition numbers: CCTCC NO, C2018159, and is named in classification as: hybridoma cell line 3H6, with a preservation date of: 2018.9.5, respectively; the anti-ASFV p30 monoclonal antibodies Mab1 and Mab2 are directed against epitopes located at p30aa145-aa154, and Mab1 and Mab2 are specifically directed against epitopes different.
The present invention claims monoclonal antibodies Mab1 and Mab2, respectively, and combinations thereof.
The invention provides a colloidal gold immunochromatographic test paper for detecting African swine fever virus antigen, which is characterized in that: comprises a PVC base plate, wherein a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed on the PVC base plate; wherein the colloidal gold pad is internally provided with a monoclonal antibody Mab1 of colloidal gold labeled anti-ASFV p30 antigen; the surface of the nitrocellulose membrane is marked with a detection line and a quality control line, the detection line is a monoclonal antibody Mab2 aiming at another epitope of the ASFV p30 antigen, and the quality control line is a goat anti-mouse IgG antibody.
anti-ASFV p30 monoclonal antibodies Mab1 and Mab2, and hybridoma cells secreting monoclonal antibody Mab1 were deposited with the Chinese type culture Collection with the addresses: wuhan university, the preservation number is: CCTCC NO, C2018158, and is named in classification as: hybridoma cell line 6G12, with a preservation date of: 2018.9.5, respectively; the hybridoma cells secreting monoclonal antibody Mab2 were deposited at the chinese type culture collection, at the address of wuhan university, with the following deposition numbers: CCTCC NO, C2018159, and is named in classification as: hybridoma cell line 3H6, with a preservation date of: 2018.9.5, respectively; the anti-ASFV p30 monoclonal antibodies Mab1 and Mab2 are directed against epitopes located at p30aa145-aa154, and Mab1 and Mab2 are specifically directed against epitopes different.
The colloidal gold labeled anti-ASFV p30 antigen monoclonal antibody Mab1 specifically comprises: labeling Mab1 with colloidal gold with a diameter of 40nm, wherein the labeling pH is 8.0, and the labeling concentration of Mab1 is 10 mug/mL; centrifuging to precipitate the colloidal gold marker, and dissolving the concentrated precipitate by using a redissolution with the pH of 8.5, wherein the redissolution consists of 10% of sucrose, 1% of PVP, 1% of BSA, 0.5% of Tween-20 and 0.1M of TrisCl, the concentration ratio of the redissolution is 125 mu L/mL, and finally, the colloidal gold compound is sprayed into a colloidal gold pad, and the spraying amount is 15 mu L/cm.
The ab2 working concentration of the detection line is 1.6mg/mL, the anti-mouse IgG antibody working concentration of the quality control line is 1.5mg/mL, the spraying amount of the Mab2 antibody and the goat anti-mouse IgG antibody during film scratching is 1 muL/cm, and the distance between the detection line and the quality control line is 0.5 cm.
A preparation method of a colloidal gold immunochromatographic test paper for detecting African swine fever virus antigen comprises the following steps:
1) preparing an SFVp30 antigen monoclonal antibody, and identifying an epitope region and an antibody subclass;
2) preparing colloidal gold immunochromatographic test paper for detecting ASFV p30 antigen.
The step 2) is specifically as follows: preparing a colloidal gold pad labeled with p30 monoclonal antibody Mab 1; spraying a nitrocellulose membrane detection line and a quality control line; and (6) assembling the test strip.
The application of the colloidal gold immunochromatographic test paper for detecting the African swine fever virus antigen in preparing the reagent for diagnosing ASFV infection, epidemiological investigation and international trade quarantine inspection of live pigs.
Specifically, the preparation method of the colloidal gold immunochromatographic test paper for detecting ASFV antigen comprises the following steps:
1. preparation of anti-ASFVp 30 antigen monoclonal antibody, epitope region and antibody subclass identification
(1) preparation of p30 recombinant antigen
Artificially synthesized codon-optimized ASFV p30 gene
catatgGACTTCATCCTGAACATCTCTATGAAAATGGAAGTTATCTTCAAAACCGACCTGCGTTCTTCTTCTCAGGTTGTTTTCCACGCTGGTTCTCTGTACAACTGGTTCTCTGTTGAAATCATCAACTCTGGTCGTATCGTTACCACCGCTATCAAAACCCTGCTGTCTACCGTTAAATACGACATCGTTAAATCTGCTCACATCTACGCTGGTCAGGGTTACACCGAACACCAGGCTCAGGAAGAATGGAACATGATCCTGCACGTTCTGTTCGAAGAAGAAACCGAATCTTCTGCTTCTTCTGAATCTATCCACGAAAAAAACGACAACGAAACCAACGAATGCACCTCTTCTTTCGAAACCCTGTTCGAACAGGAACCGTCTTCTGAAGAACCGAAAGACTCTAAACTGTACATGCTGGCTCAGAAAACCGTTCAGCACATCGAACAGTACGGTAAAGCTCCGGACTTCAACAAAGTTATCCGTGCTCACAACTTCATCCAGACCATCCACGGTACCCCGCTGAAAGAAGAAGAAAAAGAAGTTGTTCGTCTGATGGTTATCAAACTGCTGAAAAAAAAAgtcgac (SEQ ID No.1), after restriction enzyme digestion of NdeI and XhoI, inserting the restriction enzyme digestion site corresponding to plasmid pET30a (+) to construct recombinant plasmid pET-p30, transforming competent cell BLR, inoculating the recombinant bacteria carrying plasmid pET-p30 into 2 × YT culture medium containing 50 ug/mL kanamycin according to the volume ratio of 1: 100, and culturing at 37 ℃ until OD is reached600Adding 1mmol/L IPTG, and inducing at 37 deg.C for 4 h; centrifuging at 8000rpm for 2min, washing thallus with 0.01M PBS solution for 2 times, and centrifuging again; the thalli obtained by re-centrifugation is cracked by an ultrasonic instrument with the power of 50W for 10 s/time for 10 min; centrifuging at 4 deg.C and 12000rpm for 20min, and collecting precipitate; the p30 recombinant protein was purified using a Ni-NTA agarose column.
(2) Preparation of anti-p 30 monoclonal antibody
BALB/c mice, 8 weeks old, were immunized with the purified p30 recombinant protein. In the first immunization, the p30 recombinant protein and Freund's complete adjuvant are emulsified in equal volume, and mice are inoculated in the abdominal cavity, wherein each mouse contains 50 mu g p30 protein; after 7 days, the p30 recombinant protein and Freund's incomplete adjuvant are emulsified in equal volume, and mice are immunized by the second intraperitoneal inoculation way, wherein each mouse contains 50 mu g p30 protein; p30 protein was directly immunized in the third mouse abdominal cavity after 7 days, 50. mu.g/mouse; on the 3 rd day after immunization, fusion of mouse spleen cells and mouse myeloma cells SP2/0 is carried out, and HAT selective culture medium is cultured; after 10 days, the cell supernatant is detected by indirect ELISA by taking the recombinant p30 as a coating antigen, and positive hybridomas are screened.
(3) Epitope region and antibody subclass identification for anti-p 30 monoclonal antibody
According to the gene sequence of SEQ ID No.1, the following primers were synthesized:
F15’-ATCATGGACTTCATCCTG-3’(SEQ ID No.2)
R1 5’-GCAActcgagAGAAGACGGTTCCTG-3’(SEQ ID No.3)
F2 5’-ATCTGGAACATGATCCTGC-3’(SEQ ID No.4)
R2 5’-CGAActcgagTTTTTTTTTCAGCAGTTTG-3’(SEQ ID No.5)
R3 5’-CGAActcgagGATAACTTTGTTGAAGTCC-3’(SEQ ID No.6)
R4 5’-CGAActcgagGGTTTTCTGAGCCAGCATG-3’(SEQ ID No.7)
R5 5’-CGAActcgagGTCTTTCGGTTCTTCAGAAG-3’(SEQ ID No.8)
R6 5’-CGAActcgag AGCTTTACCGTACTGTTCG-3’(SEQ ID No.9)
using pET-p30 plasmid as template, respectively using primers F1/R1, F2/R2, F1/R3, F1/R4, F1/R5 and F1/R6 to PCR amplify corresponding DNA fragment, using DNA polymerase
HS DNA polymerase, after the PCR product is cut by restriction enzyme XhoI, inserting into plasmid pET30a (+) EcoRV and XhoI cutting sites, respectively transforming the constructed recombinant plasmids into competent cells BLR, and inducing the inserted genes of the recombinant bacteria by using the method; bacteria are lysed, after SDS-PAGE electrophoresis, the bacterial proteins are transferred to a nitrocellulose membrane, an immunoblotting experiment is carried out, and the epitopes aimed by the screened monoclonal antibodies Mab1 and Mab2 are both located at p30aa145-aa 154. And (3) taking the recombinant p30 as a coating antigen, calculating an Additive Index (AI) by adopting an indirect ELISA method additive test, and determining that the monoclonal antibodies Mab1 and Mab2 respectively aim at different antigen epitopes.
The obtained monoclonal antibody subclasses were identified by using a mouse monoclonal antibody Ig class/subclass identification ELISA kit, and it was determined that monoclonal antibodies Mab1 and Mab2 were both mouse IgG1 subclass antibodies and light chains were both kappa chains.
(4) Preparation of Mab1 and Mab2 ascites
Injecting 0.5mL of sterile liquid paraffin into the abdominal cavity of a BALB/c mouse aged 8 weeks; after 1 week, the mice were injected with hybridoma cells secreting Mab1 and Mab2, 106Collecting ascites after 6 days, centrifuging at 12000rpm to remove cells and fat, purifying with Protein G affinity chromatography column, centrifuging in ultrafiltration centrifuge tube to remove salt, re-dissolving with equal volume of 0.005% NaCl solution (pH7.0), and storing in refrigerator at-70 deg.C.
2. Preparation of colloidal gold immunochromatographic test paper for detecting ASFV p30 antigen
(1) Preparation of colloidal gold pad labeled with p30 monoclonal antibody Mab1
The Mab1 ascites is labeled with 40nm colloidal gold Mab1, pH8.0 when labeled, and Mab1 labeled concentration is 10 mug/mL; the colloidal gold-labeled substance was precipitated by centrifugation, and the precipitate was dissolved and concentrated in a reconstitution solution (10% sucrose, 1% PVP, 1% BSA, 0.5% Tween-20, 0.1M Tris Cl, pH 8.5) at a concentration ratio of 125. mu.L/mL. And spraying the concentrated colloidal gold compound into a colloidal gold pad by using a gold spraying and membrane scratching all-in-one machine, wherein the spraying amount is 15 mu L/cm, and naturally drying.
(2) Nitrocellulose membrane detection line and quality control line spraying
Spraying a detection line (T) and a quality control line (C) on the surface of the nitrocellulose membrane by using a gold spraying and membrane scribing integrated machine, wherein the detection line is a monoclonal antibody Mab2 aiming at ASFVp30 antigen, and the quality control line is a goat anti-mouse IgG antibody; the working concentration of Mab2 is 1.6mg/mL, the working concentration of goat anti-mouse IgG antibody is 1.5mg/mL, the spraying amount is 1 muL/cm when the two are scratched, and the distance between the detection line and the quality control line is 0.5 cm.
(3) Test strip assembly
The sticking surface of the PVC bottom plate is upward, and the nitrocellulose membrane is stuck in the middle of the PVC plate; then, before the gold label pad is adhered to the nitrocellulose membrane, the gold label pad and the nitrocellulose membrane are overlapped by 0.2 cm; and respectively adhering a sample pad and a water absorption pad on the PVC bottom plate in front of the gold-labeled pad and behind the nitrocellulose membrane, wherein the sample pad and the water absorption pad are respectively overlapped with the gold-labeled pad and the nitrocellulose membrane by 0.2 cm. The assembled PVC plate and the attachment are cut into test strips with the width of 0.4cm by a slitter and then are put into a plastic card.
The invention is not limited to the antigen produced by ASFV detected by the detection method, but also includes other double-antibody sandwich colloidal immunochromatography techniques which can be used for ASFV detection. The antibody label can be replaced by other labeling methods such as enzyme label, and the sequences of the recombinant antigen and the monoclonal antibody and the targeted virus protein can be replaced by other sequences and the targeted virus protein can be different virus proteins.
The colloidal gold immunochromatographic test paper for detecting the ASFV antigen is suitable for detecting peripheral blood mononuclear cells of pigs, lymph nodes of the pigs and p30 antigen in ASFV in-vitro infected cells, can diagnose the ASFV infection in a short time, and is particularly suitable for on-site ASFV infection diagnosis, epidemiological investigation and international trade quarantine inspection of live pigs.
Drawings
FIG. 1 schematic representation of the identification of Mab1 against p30 epitope;
m, protein molecule quality standard, 1, p30 aa1-aa128, 2, p30 aa125-aa194, 3, p30 aa1-aa161, 4, p30 aa1-aa144, 5, p30 aa1-aa134, 6, p30 aa1-aa 154;
FIG. 2 schematic representation of the identification of Mab2 against p30 epitope;
m, protein molecule quality standard, 1, p30 aa1-aa128, 2, p30 aa125-aa194, 3, p30 aa1-aa161, 4, p30 aa1-aa144, 5, p30 aa1-aa134, 6, p30 aa1-aa 154;
FIG. 3 is a schematic diagram of the assembly of the gold immunochromatographic test strip;
A. schematic diagram of the right side inside the colloidal gold immunochromatographic test paper; B. the front view inside the colloidal gold immunochromatographic test paper is schematic; C. packing the colloidal gold immunochromatographic test paper with a plastic shell;
1, a PVC bottom plate, 2, a water absorption pad, 3, a nitrocellulose membrane, 4, a colloidal gold pad, 5, a sample pad, 6, a plastic shell, 7, a quality control line, 8, a detection line and 9, a sample adding hole;
FIG. 4 is a graph showing the result of the lowest detection sensitivity of the colloidal gold immunochromatographic test strip;
FIG. 5 is a graph showing the results of detection of p30 antigen in ASFV in vitro infected cells using colloidal gold immunochromatographic strip;
FIG. 6 is a diagram showing the results of a test for detecting specificity with colloidal gold immunochromatographic test paper;
FIG. 7 is a diagram showing the results of a field serum sample test using a colloidal gold immunochromatographic test strip;
FIG. 8 is a diagram showing the results of a field tissue sample test using a colloidal gold immunochromatographic test strip;
hybridoma cells secreting monoclonal antibody Mab1 were deposited with the chinese type culture collection, address: wuhan university, the preservation number is: CCTCC NO, C2018158, and is named in classification as: hybridoma cell line 6G12, with a preservation date of: 2018.9.5, respectively; the hybridoma cells secreting monoclonal antibody Mab2 were deposited at the chinese type culture collection, at the address of wuhan university, with the following deposition numbers: CCTCC NO, C2018159, and is named in classification as: hybridoma cell line 3H6, with a preservation date of: 2018.9.5.
Detailed Description
For the purpose of illustrating the technical solutions and technical objects of the present invention, the present invention will be further described with reference to the following description and specific embodiments.
The source of the biological material is as follows:
coli expression vector pET30a (+): purchased from Novogen corporation (Cat No. 69909.3);
eukaryotic expression vector pcDNA3.0: purchased from Invitrogen corporation;
ASFV p30 eukaryotic expression vector pcDNA-p 30: constructing the laboratory;
escherichia coli (e.coli) BLR competent cells: purchased from Beijing Huayue ocean organisms (Cat No, C345001);
PK15 cells, Marc-145 cells, SP2/0 cells: storing in the laboratory;
Goat Anti-mouse IgG H&L(Alexa
488) i.e., goat anti-mouse IgG: purchased from Abcam (CatNo, ab 150169);
ASFVGenotype II (Russian) infected COS-1 cells: provided by the Russian Federal virology and microbiology research center;
porcine pseudorabies virus (PRV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) attenuated vaccine: purchased from guangdong Yongshun biopharmaceutical, Inc.;
porcine circovirus type 2 (PCV-2): infectious disease university of Yangzhou university graduate culture room high stem leaf professor gives a gift;
hog cholera virus (CSFV) T strain: purchased from the institute of veterinary medicine in China.
Example 1 preparation of colloidal gold immunochromatographic test paper for detecting ASFV antigen
Preparation of p30 recombinant antigen
Artificially synthesized and codon-optimized ASFV p30 gene (SEQ ID No.1) is inserted into plasmid pET30a (+), recombinant plasmid pET-p30 is constructed, competent cell BLR is transformed, the recombinant bacterium carrying plasmid pET-p30 is inoculated into 2 × YT culture medium containing 50 mu g/mL kanamycin according to the volume ratio of 1: 100, and the culture is carried out at 37 ℃ until OD600Adding 1mmol/L IPTG, and inducing at 37 deg.C for 4 h; centrifuging at 8000rpm for 2min to obtain thallusWashing with 0.01M PBS solution for 2 times, and centrifuging again; the thalli obtained by re-centrifugation is cracked by an ultrasonic instrument with the power of 50W for 10 s/time for 10 min; centrifuging at 4 deg.C and 12000rpm for 20min, and collecting precipitate; the p30 recombinant protein was purified using a Ni-NTA agarose column.
2. Preparation of anti-p 30 monoclonal antibody
BALB/c mice, 8 weeks old, were immunized with the purified p30 recombinant protein. In the first immunization, the p30 recombinant protein and Freund's complete adjuvant are emulsified in equal volume, and mice are inoculated in the abdominal cavity, and 50 mu gp30 protein/mouse; after 7 days, the p30 recombinant protein and Freund's incomplete adjuvant are emulsified in equal volume, and the mice are immunized by the second intraperitoneal inoculation way, and 50 mu gp30 protein/mouse; p30 protein was directly immunized in the third mouse abdominal cavity after 7 days, 50. mu.g/mouse; on the 3 rd day after immunization, fusion of mouse spleen cells and mouse myeloma cells SP2/0 is carried out, and HAT selective culture medium is cultured; after 10 days, the cell supernatant is detected by indirect ELISA by taking the recombinant p30 as a coating antigen, and positive hybridomas are screened.
3. Epitope region and antibody subclass identification for anti-p 30 monoclonal antibody
According to the gene sequence of SEQ ID No.1, the following primers were synthesized:
F1 5’-ATCATGGACTTCATCCTG-3’(SEQ ID No.2)
R1 5’-GCAActcgagAGAAGACGGTTCCTG-3’(SEQ ID No.3)
F2 5’-ATCTGGAACATGATCCTGC-3’(SEQ ID No.4)
R2 5’-CGAActcgagTTTTTTTTTCAGCAGTTTG-3’(SEQ ID No.5)
R3 5’-CGAActcgagGATAACTTTGTTGAAGTCC-3’(SEQ ID No.6)
R4 5’-CGAActcgagGGTTTTCTGAGCCAGCATG-3’(SEQ ID No.7)
R5 5’-CGAActcgagGTCTTTCGGTTCTTCAGAAG-3’(SEQ ID No.8)
R6 5’-CGAActcgag AGCTTTACCGTACTGTTCG-3’(SEQ ID No.9)
using pET-p30 plasmid as template, respectively using primers F1/R1(p30 aa1-aa128), F2/R2(p30 aa125-aa194), F1/R3(p30 aa1-aa161), F1/R4(p30 aa1-aa144), F1/R5(p30 aa1-aa134), F1/R6(p30 aa1-aa154) to PCR amplify corresponding DNA fragment,DNA polymerase used
HS DNA polymerase (Takara Bio-engineering Co., Ltd.), after the PCR product is cut by restriction enzyme XhoI, inserting into plasmid pET30a (+) EcoRV and XhoI cutting sites, respectively transforming the constructed recombinant plasmids into competent cells BLR, and inducing the inserted genes of the recombinant bacteria by using the method; bacteria are lysed, after SDS-PAGE electrophoresis, the bacterial proteins are transferred to a nitrocellulose membrane, an immunoblotting experiment is carried out, and the epitopes aimed by the screened monoclonal antibodies Mab1 and Mab2 are both positioned at p30aa145-aa154 (FIGS. 1 and 2). And (3) taking the recombinant p30 as a coating antigen, calculating an Additive Index (AI) by adopting an indirect ELISA method additive test, and determining that the monoclonal antibodies Mab1 and Mab2 respectively aim at different antigen epitopes.
The obtained monoclonal antibody subclass was identified by using a mouse monoclonal antibody Ig class/subclass identification ELISA kit (Shanghai Fengshi Biotech Co., Ltd.), and it was confirmed that both monoclonal antibodies Mab1 and Mab2 were mouse IgG1 subclass antibodies and light chains were kappa chains.
Preparation of Mab1 and Mab2 ascites
Injecting 0.5mL of sterile liquid paraffin into the abdominal cavity of a BALB/c mouse aged 8 weeks; after 1 week, the mice were injected with hybridoma cells secreting Mab1 and Mab2, 106Ascites was collected 6 days later, centrifuged at 12000rpm to remove cells and fat, purified by Protein G affinity column (GE Co.), centrifuged at 6000rpm in an ultrafiltration centrifuge tube (Millipore) for 30min to remove salt, redissolved with an equal volume of 0.005% NaCl solution (pH7.0), stored in a refrigerator at-70 ℃ and ready for labeling and streaking of colloidal gold below.
5. Preparation of colloidal gold pad labeled with p30 monoclonal antibody Mab1
(1) Optimal pH determination of Mab 1-labeled colloidal gold: 1mL of colloidal gold solution (Weifang Bainuoddi Biotechnology Co., Ltd.) with a diameter of 40nm was dispensed into 1.5mL finger tube, and 9 tubes were filled with 0.2mol/L hydrochloric acid solution or 0.2mol/L K mol2CO3The solution, the precision pH paper determines the pH value of the colloidal gold solution, so that the pH value of the solution in the finger-shaped tube is respectively pH5.0, pH5.5, pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5 and pH9.0 in sequence; addingAdding 20 mu g of ASFV p54 recombinant protein, mixing uniformly and standing for 1 h; then 100 mul of 10% NaCl is added, and the result is observed after standing for 10min, and the color of the solution is unchanged, namely the optimum pH value of the colloidal gold labeled antigen. The optimum pH value of the Mab1 labeled colloidal gold was determined to be 8.0.
(2) And (3) determining the optimal labeling concentration of Mab1 and colloidal gold: 0.005mol/L NaCl solution to adjust the concentration of Mab1 to 200. mu.g/mL, and adding 10. mu.L, 20. mu.L, 30. mu.L, 40. mu.L, 50. mu.L, 60. mu.L, 70. mu.L, 80. mu.L, 90. mu.L of recombinant protein solution and 0.005mol/L NaCl solution into a finger-shaped tube containing 1mL of colloidal gold; according to the above results, 0.2mol/L K was added2CO3Adjusting to the optimal pH value, uniformly mixing and standing for 1 h; and then adding 100 mu L of 10% NaCl into each tube, standing for 10min, observing the color change of the colloidal gold solution, and if the color does not change, adding the minimum amount of recombinant protein to stabilize the colloidal gold. The optimal labeling concentration of Mab1 and colloidal gold was determined to be 10. mu.g/mL.
(3) Mab1 colloidal gold labeling: with 0.2mol/L K2CO3Adjusting the pH value of the colloidal gold solution to 8.0, adding a proper amount of 200 mu g/mL Mab1 solution to a final concentration of 10 mu g/mL, and stirring for 30 min; slowly adding appropriate amount of 10% BSA to make its final concentration 1%, stirring for 30 min; then centrifuging the solution at 1000rpm and 4 ℃ for 15min, and taking the supernatant; the supernatant was centrifuged at 12000rpm at 4 ℃ for 30min, the supernatant was discarded, and the precipitate was dissolved in a reconstitution solution (10% sucrose, 1% PVP, 1% BSA, 0.5% Tween-20, 0.1M Tris Cl, pH 8.5) at a concentration ratio of 125. mu.L/mL to concentrate the precipitate. Spraying the concentrated colloidal gold compound into a colloidal gold pad (AlhStorm 8964) by using a Biodot XYZ3060 gold spraying and film scratching integrated machine, wherein the spraying amount is 15 mu L/cm, and naturally drying.
6. Nitrocellulose membrane detection line and quality control line spraying
Spraying a detection line (T) and a quality control line (C) on the surface of a nitrocellulose membrane (Sartorius CN140) by using a gold spraying and membrane scribing integrated machine, wherein the detection line is a monoclonal antibody Mab2 aiming at ASFVp30 antigen, and the quality control line is a goat anti-mouse IgG antibody; the working concentration of Mab2 is 1.6mg/mL, the working concentration of goat anti-mouse IgG antibody is 1.5mg/mL, the spraying amount is 1 muL/cm when the two are scratched, and the distance between the detection line and the quality control line is 0.5 cm.
7. Colloidal gold immunochromatographic test paper assembly
(1) A PVC base plate with a width of 8cm and an upward adhesive surface (Weifang Bainuoddi Biotechnology Co., Ltd.) was adhered to the middle of the PVC base plate with a width of 2.5 cm;
(2) a 0.5cm wide gold-labeled pad (AlhStorm 8964) was attached to the nitrocellulose membrane, overlapping the solid phase nitrocellulose membrane by 0.2 cm;
(3) a2.7 cm wide sample pad (Weifang Bainuoddi Biotech Co., Ltd.) was attached to the front of the gold-labeled pad, overlapping the gold-labeled pad by 0.2 cm;
(4) a wicking pad (Weifang Bainuodai Biotechnology Co., Ltd.) 3.2cm wide was attached to the nitrocellulose membrane and overlapped 0.2cm with the solid nitrocellulose membrane;
(5) a microcomputer automatic cutting machine (Shanghai gold Co., Ltd., ZQ2000) cuts into strips of 4mm in width;
(6) the strips were fitted with a protective plastic housing (Weifang Bainuoddi Biotech Co., Ltd.).
8. Colloidal gold immunochromatographic test paper result judgment
If red strips appear on the detection line (T) and the quality control line (C), the ASFV antigen in the sample to be detected is judged to be positive; if the quality control line (C) has a red strip and the detection line (T) has no red strip, determining that the ASFV antigen in the sample to be detected is negative; if there is no red band at the control line (C), the test strip is disabled. The structure and appearance of the assembled test strip is shown in fig. 3.
Example 2 determination of minimum detection sensitivity of colloidal gold immunochromatographic strip
The purified recombinant protein p30 was diluted to 943. mu.g/mL, 9.43. mu.g/mL, 94.3ng/mL, and 0.943ng/mL, and 85. mu.L of each dilution was aspirated, added to the wells of a test paper, and the results were observed within 5 min. The results show that red strips can appear on the quality control line and the detection line when four samples with different concentrations are detected, and the color of the detection line is slightly lighter when the samples with the concentration of 0.943ng/mL are detected (figure 4), which indicates that the colloidal gold immunochromatographic test paper can detect 80pg of ASFV p30 protein at the lowest.
Example 3 detection of ASFV p30 antigen in porcine blood cells by colloidal gold immunochromatographic strip
Transfection of PK15 cells with pcDNA-p30 plasmid
Mu.g pcDNA-p30 eukaryotic expression plasmid, 9. mu.L Lipofectamine2000(Invitrogen) were dissolved in 500. mu.L Opti-MEM medium (Gibco), and the mixture was allowed to stand at room temperature for 20min, and then allowed to stand at room temperature for 5 min; after discarding 95% PK15 cell culture medium grown in 6-well cell culture plates (Corning), and washing with sterile PBS, the transfection mixture was added thereto at 37 ℃ with 5% CO2Standing in an incubator for 4 h; the transfection mixture was discarded, changed to DMEM medium (Gibco) containing 1% fetal bovine serum, PK15 cells were trypsinized after 48h, the cells were pelleted by centrifugation, sterilized PBS was suspended, and pcDNA3.0 empty vector transfection control group was established.
Taking a small amount of suspended cells, adding 8% paraformaldehyde with the same volume for fixing for 10min, washing the cells with PBS, and centrifuging and precipitating the cells; adding anti-ASFV p30 monoclonal antibody Mab1, acting at 37 ℃ for 30min, washing cells for 3 times by PBS, and centrifuging to precipitate cells; add Goat Anti-mouse IgG H diluted to working concentration&L(Alexa
488) Fluorescent antibody, acting at 37 ℃ for 30min, washing cells for 3 times by PBS; the transfection efficiency of the cells was determined to be 17.93% by flow cytometry (CyAn ADP 7).
2. Detection of PK15 intracellular ASFV p30 antigen by colloidal gold immunochromatographic test paper
After PK15 transfected cells are counted, the cells are centrifugally precipitated, supernatant is discarded, 1% NP-40 (prepared by 0.01 MpH7.4PBS) cell lysate is added to fully lyse the cells, the cells are diluted by 10 times of gradient of the cell lysate, 85 mu L of cell lysate is respectively absorbed, a colloidal gold immunochromatographic test paper detection hole is added, and the observation result in 5min is shown in Table 12And (3) expressing ASFV p30 cells.
TABLE 1 results of detecting ASFV p30 in pig cells by using colloidal gold immunochromatographic test paper
3. Detection of ASFV p30 antigen in pig blood cells by colloidal gold immunochromatographic test paper
(1) Collecting pig blood, adding 1/10 volume of 3.8% sodium citrate solution, and mixing; 300 mu L of the anticoagulation blood is respectively added into 4 centrifuge tubes with the volume of 1.5 mL; different numbers of PK15 cells as shown in table 2 were added to the centrifuge tubes.
(2) Centrifuging at 3000rpm for 2min, discarding the plasma supernatant, adding 900. mu.L of RBC lysate (0.16M NH)4Cl,0.13mM EDTA,12mM NaHCO3) After the mixture was inverted and mixed, the mixture was left standing at room temperature for 5 min.
(3) Centrifuging at 3000rpm for 2min, and discarding erythrocyte lysis supernatant.
(4) Adding 85 μ L of karyocyte lysate (1% NP-40), blowing and sucking by a pipette, adding colloidal gold immunochromatographic test paper sample adding holes, and observing the result within 5 min.
TABLE 2 detection results of PK15 cells added with expression p30 in porcine blood cells
As a result, the colloidal gold immunochromatographic test strip can detect 9.1 × 10 in blood cells at the lowest3And (3) expressing ASFV p30 cells.
Example 4 detection of p30 antigen in ASFV in vitro infected cells by colloidal gold immunochromatographic strip
COS-1 cells are infected by ASFVGenotype II (Russian) in a 6-hole cell culture plate, cell culture supernatant is sucked out after 48 hours, 3mL of 1% NP-40 cell lysate is added, a pipette is used for sucking the lysed cells, 85 mu L of cell lysate is sucked, colloidal gold immunochromatographic test paper is added into sample adding holes, and results are observed within 5 min. The test result showed positive (fig. 5).
EXAMPLE 5 specificity test by colloidal gold immunochromatographic test strip
Respectively inoculating PRV, PCV-2 and CSFV T strains into PK15 cells growing to 95% in a 24-hole cell culture plate, and inoculating PRRSV into Marc-145 cells growing to 90% in the 24-hole cell culture plate; sucking out cell culture supernatant after 48h, adding 200 μ L of 1% NP-40 cell lysate, sucking the lysed cells by a pipette, sucking 85 μ L of cell lysate, adding a sample hole of colloidal gold immunochromatographic test paper, and observing the result within 5 min. The test result showed negative (fig. 6).
Example 6 field sample test on colloidal gold immunochromatographic test strip
4 parts of pig whole blood (the qPCR detection shows that the nucleic acid of the ASFV is positive), 50 microliter of serum is taken, 50 microliter of 1% NP-40 cell lysate is added, a pipettor blows and sucks the lysed cells, 85 microliter of cell lysate is sucked, a colloidal gold immunochromatographic test paper sample adding hole is added, the result is observed within 5min, and the detection result shows that the test is positive (figure 7); 4 parts of pig lung, spleen, kidney and tonsil (the qPCR detection shows that ASFV nucleic acid is negative), 200 mug of the tissues are respectively added with 1mL of sterile PBS, after grinding, 50 mug of supernatant is sucked, 50 mug of 1% NP-40 cell lysate is added, a pipette is used for sucking and cracking cells, 85 mug of cell lysate is sucked, a sample adding hole of a colloidal gold immunochromatographic test paper is added, and the detection result shows that the cells are negative within 5min (figure 8).
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
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Claims (7)
1. A colloidal gold immunochromatographic test paper for detecting African swine fever virus antigen is characterized in that: comprises a PVC base plate, wherein a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad are sequentially fixed on the PVC base plate; wherein the colloidal gold pad is internally provided with a monoclonal antibody Mab1 of colloidal gold labeled anti-ASFV p30 antigen; the surface of the nitrocellulose membrane is marked with a detection line and a quality control line, the detection line is a monoclonal antibody Mab2 aiming at another epitope of ASFV p30 antigen, and the quality control line is a goat anti-mouse IgG antibody; anti-ASFV p30 monoclonal antibodies Mab1 and Mab2, and hybridoma cells secreting monoclonal antibody Mab1 were deposited at the university of Wuhan collection, at the address: wuhan university, the preservation number is: CCTCC NO, C2018158, and is named in classification as: hybridoma cell line 6G12, with a preservation date of: 2018.9.5, respectively; the hybridoma cells secreting monoclonal antibody Mab2 were deposited at the university of wuhan collection, at the address china, wuhan university, with the following deposition numbers: CCTCC NO, C2018159, and is named in classification as: hybridoma cell line 3H6, with a preservation date of: 2018.9.5, respectively; the anti-ASFV p30 monoclonal antibodies Mab1 and Mab2 are directed against epitopes located at p30aa145-aa154, and Mab1 and Mab2 are specifically directed against epitopes different.
2. The African swine fever virus antigen colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the colloidal gold labeled anti-ASFV p30 antigen monoclonal antibody Mab1 specifically comprises: labeling Mab1 with colloidal gold with a diameter of 40nm, wherein the labeling pH is 8.0, and the labeling concentration of Mab1 is 10 mug/mL; centrifuging to precipitate the colloidal gold marker, and dissolving the concentrated precipitate by using a redissolution with the pH of 8.5, wherein the redissolution consists of 10% of sucrose, 1% of PVP, 1% of BSA, 0.5% of Tween-20 and 0.1M of TrisCl, the concentration ratio of the redissolution is 125 mu L/mL, and finally, the colloidal gold compound is sprayed into a colloidal gold pad, and the spraying amount is 15 mu L/cm.
3. The African swine fever virus antigen colloidal gold immunochromatographic test strip according to claim 1, which is characterized in that: the working concentration of the Mab2 of the detection line is 1.6mg/mL, the working concentration of the anti-mouse IgG antibody of the quality control line is 1.5mg/mL, the spraying amount of the Mab2 antibody and the goat anti-mouse IgG antibody during film scratching is 1 muL/cm, and the distance between the detection line and the quality control line is 0.5 cm.
4. The method for preparing the African swine fever virus antigen colloidal gold immunochromatographic test strip according to any one of claims 1 to 3, comprising the steps of:
1) preparing an ASFVp30 antigen monoclonal antibody, and identifying an epitope region and an antibody subclass;
2) preparing colloidal gold immunochromatographic test paper for detecting ASFV p30 antigen.
5. The preparation method of the African swine fever virus antigen colloidal gold immunochromatographic test paper according to claim 4, wherein the step 2) is specifically as follows: preparing a colloidal gold pad labeled with p30 monoclonal antibody Mab 1; spraying a nitrocellulose membrane detection line and a quality control line; and (6) assembling the test strip.
6. Use of the African swine fever virus antigen colloidal gold immunochromatographic strip of any one of claims 1-4 in the preparation of reagents for ASFV infection diagnosis, epidemiological investigation and swine international trade quarantine inspection.
7. anti-ASFV p30 monoclonal antibodies Mab1 and Mab2, characterized in that the hybridoma cells secreting monoclonal antibody Mab1 are deposited with the university of Wuhan collection, at the address: wuhan university, the preservation number is: CCTCC NO, C2018158, and is named in classification as: hybridoma cell line 6G12, with a preservation date of: 2018.9.5, respectively; the hybridoma cells secreting monoclonal antibody Mab2 were deposited at the university of wuhan collection, at the address china, wuhan university, with the following deposition numbers: CCTCC NO, C2018159, and is named in classification as: hybridoma cell line 3H6, with a preservation date of: 2018.9.5, respectively; the anti-ASFV p30 monoclonal antibodies Mab1 and Mab2 are directed against epitopes located at p30aa145-aa154, and Mab1 and Mab2 are specifically directed against epitopes different.
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| CN109813904A (en) * | 2019-03-27 | 2019-05-28 | 丁建红 | African swine fever virus rapid detection card |
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| CN111781363A (en) * | 2020-08-12 | 2020-10-16 | 江苏省农业科学院 | Quantum dot microsphere immunochromatography test strip for detecting mucosa sIgA antibody of African swine fever virus and application thereof |
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