CN115947795A - Recombinant protein for detecting ASFV antibody, double-antigen sandwich ELISA kit and application thereof - Google Patents
Recombinant protein for detecting ASFV antibody, double-antigen sandwich ELISA kit and application thereof Download PDFInfo
- Publication number
- CN115947795A CN115947795A CN202211064046.0A CN202211064046A CN115947795A CN 115947795 A CN115947795 A CN 115947795A CN 202211064046 A CN202211064046 A CN 202211064046A CN 115947795 A CN115947795 A CN 115947795A
- Authority
- CN
- China
- Prior art keywords
- asfv
- antibody
- recombinant protein
- recombinant
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000701386 African swine fever virus Species 0.000 title claims abstract description 84
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 66
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 66
- 239000000427 antigen Substances 0.000 title claims abstract description 48
- 238000003118 sandwich ELISA Methods 0.000 title claims abstract description 29
- 210000002966 serum Anatomy 0.000 claims abstract description 93
- 239000000243 solution Substances 0.000 claims abstract description 40
- 238000002965 ELISA Methods 0.000 claims abstract description 36
- 238000001514 detection method Methods 0.000 claims abstract description 30
- 102000004190 Enzymes Human genes 0.000 claims abstract description 25
- 108090000790 Enzymes Proteins 0.000 claims abstract description 25
- 241000282898 Sus scrofa Species 0.000 claims abstract description 24
- 102000036639 antigens Human genes 0.000 claims abstract description 23
- 108091007433 antigens Proteins 0.000 claims abstract description 23
- 101900020862 African swine fever virus Inner membrane protein p54 Proteins 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000013642 negative control Substances 0.000 claims abstract description 20
- 239000013641 positive control Substances 0.000 claims abstract description 19
- 238000005406 washing Methods 0.000 claims abstract description 19
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000012089 stop solution Substances 0.000 claims abstract description 8
- 239000003085 diluting agent Substances 0.000 claims abstract description 7
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 7
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 7
- 239000013612 plasmid Substances 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 244000309466 calf Species 0.000 claims description 10
- 108091008146 restriction endonucleases Proteins 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 8
- 241000282887 Suidae Species 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000013598 vector Substances 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 208000007407 African swine fever Diseases 0.000 abstract description 18
- 108090000623 proteins and genes Proteins 0.000 abstract description 14
- 239000007788 liquid Substances 0.000 abstract description 10
- 102000004169 proteins and genes Human genes 0.000 abstract description 9
- 239000012295 chemical reaction liquid Substances 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 208000015181 infectious disease Diseases 0.000 abstract description 3
- 229940031626 subunit vaccine Drugs 0.000 abstract description 3
- 108010042365 Virus-Like Particle Vaccines Proteins 0.000 abstract description 2
- 238000011841 epidemiological investigation Methods 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 230000000405 serological effect Effects 0.000 abstract description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 10
- 230000003053 immunization Effects 0.000 description 8
- 238000002649 immunization Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 238000011049 filling Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 241000283707 Capra Species 0.000 description 4
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 4
- 241000702619 Porcine parvovirus Species 0.000 description 4
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 4
- 239000002250 absorbent Substances 0.000 description 4
- 230000002745 absorbent Effects 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000011888 foil Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 238000003119 immunoblot Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 241001673669 Porcine circovirus 2 Species 0.000 description 2
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 101150049619 p30 gene Proteins 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 238000010023 transfer printing Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of biology, in particular to a recombinant protein for detecting African Swine Fever Virus (ASFV) antibody, a double-antigen sandwich ELISA kit and application thereof, wherein the kit comprises an antigen-coated ELISA plate, an enzyme-labeled antibody, african swine fever positive control serum, ASF negative control serum, diluent, reaction liquid, 10 x washing liquid, TMB substrate solution and stop solution. The antigen of the enzyme label plate in the kit is recombinant ASFV p54 and p30 fusion protein (p 54-30-His) carrying His label. The kit can be used for serological diagnosis of ASFV infection, antibody level monitoring, epidemiological investigation and the like, can effectively avoid the interference of the tag protein when the recombinant subunit vaccine or the virus-like particle vaccine is used for immune pig antibody detection, and provides an accurate and effective method for ASFV antibody detection.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant protein for detecting an ASFV antibody, a double-antigen sandwich ELISA kit and application thereof.
Background
African Swine Fever (ASF) is a highly contagious, febrile infectious disease of domestic or wild pigs caused by African Swine Fever Virus (ASFV), and highly pathogenic strains may cause 100% of the deaths of infected pigs.
Currently available diagnostic techniques for ASF mainly include three major categories: nucleic acid detection, antigen detection and antibody detection. Virulent strains often cause acute death of pigs, and the antibody detection difficulty is high; after the medium and low toxicity strains are infected, the disease course is longer, the body can be stimulated to generate better humoral immune response, antibody detection is one of effective diagnosis methods, OIE also clearly indicates that the recovered pig or the pig infected for a period of time has ASFV antibodies in vivo, and whether the ASFV is infected or not can be confirmed through antibody detection.
The ASFV antibody detection method mainly takes ELISA as a main part, and comprises indirect ELISA, blocking ELISA/competitive ELISA and the like. The indirect ELISA is coated by the recombinant antigen, the detection sensitivity is high, the preparation of the kit is relatively simple, but fusion tag protein (such as His tag) is used during the purification of the recombinant antigen, while virus-like particle (VLPs) subunit vaccine used in the clinical pig is prepared by using the His tag purified antigen, so that a certain level of tag protein antibody exists in the normal pig body after multiple immunizations, and although the serum is greatly diluted during the detection, false positive of the detection result is easy to appear; blocking ELISA/competitive ELISA requires the use of an enzyme-labeled specific antibody, ASFV antibody in pig serum is combined with antigen on an ELISA plate to block adsorption of the enzyme-labeled antibody, and the negative and positive results are determined by substrate solution color development.
Disclosure of Invention
In order to realize the aim, the invention uses recombinant protein p54-30-His to coat an ELISA plate, adsorbs anti-ASFV p54 and p30 antibodies of a sample variety to be detected through antigen-antibody reaction, adds a reaction solution, indirectly adsorbs the antibodies and the recombinant protein GST-p54-30, utilizes HRP-marked anti-GST enzyme-labeled antibodies to combine with GST antigens adsorbed on the ELISA plate, compares the substrate solution with a critical value after color development, judges whether ASFV antibodies exist in a sample to be detected, and establishes and assembles a double-antigen sandwich ELISA kit for detecting the ASFV antibodies based on the principle. The kit can be used for serological diagnosis of ASFV infection, antibody level monitoring, epidemiological investigation and the like, can effectively avoid the interference of the tag protein when recombinant subunit vaccine or virus-like particle (VLPs) vaccine is used for immune pig antibody detection, and provides an accurate and effective method for ASFV antibody detection.
The technical scheme adopted by the invention is as follows:
a recombinant protein for detecting an ASFV antibody takes ASFV p54 and p30 as antigens, the recombinant protein sequence is shown as SEQ ID No.1, and the ASFV antigen amino acid sequence is shown as SEQ ID No. 2.
The invention also provides a preparation method of the recombinant protein for detecting the ASFV antibody, which comprises introducing restriction enzyme sites NdeI and XhoI into both ends of p54 and p30 genes respectively, and inserting the synthesized recombinant protein into a vector pUC 57.
A double-antigen sandwich ELISA kit for detecting ASFV antibody is characterized by comprising the recombinant protein for detecting the ASFV antibody, an ELISA plate, an HRP-labeled anti-GST enzyme antibody, ASF positive control serum, ASF negative control serum, diluent, reaction liquid, 10 Xwashing liquid, TMB substrate solution and stop solution.
Furthermore, the antigen of the 96-well enzyme label plate coated in the kit is recombinant ASFV p54 carrying His label, p30 recombinant protein p54-30-His; the reaction solution is recombinant ASFV p54 and p30 recombinant protein GST-p54-30 with working concentration and carrying GST tags.
Furthermore, the recombinant protein p54-30-His is obtained by expression and purification in BL21 (DE 3) escherichia coli by using a plasmid pET-30a (+) loaded with the recombinant protein; the recombinant protein GST-P54-30 is obtained by expressing and purifying plasmid pGEX-6P-1 loaded with the recombinant protein in BL21 (DE 3) escherichia coli.
Further, the HRP-labeled anti-GST enzyme-labeled antibody is an HRP-labeled mouse anti-GST monoclonal antibody; the ASF positive control serum is prepared from recombinant p54 and p30 fusion protein immune pigs; the ASF negative control serum is healthy pig serum.
Further, the dilution was 5% calf serum containing 0.01% proclin300;
the 10 × washing solution is: naCl 80g, KCl 2g, na 2 HPO 4 14.4g，KH 2 PO 4 2.4g, tween-20.5 mL, proclin 300.1mL, dissolving in 800mL of distilled water, adjusting pH to 7.4, fixing the volume to 1L, filtering and sterilizing by a 0.22 mu m filter, and storing at 4 ℃;
the stop solution is 2 MH 2 SO 4 . The double-antigen sandwich ELISA kit for detecting the ASFV antibody comprises a 96-hole ELISA plate for coating recombinant ASFV p54 and p30 fusion proteins carrying His labels, reaction liquid of the recombinant ASFV p54 and p30 fusion proteins carrying GST labels, an enzyme-labeled antibody of an HRP-labeled anti-GST monoclonal antibody, ASFV positive control serum prepared by a recombinant p54 and p30 fusion protein immune pig and ASFV negative control serum from a healthy pig.
The invention also provides a preparation method of the double-antigen sandwich ELISA kit for detecting ASFV antibody, which is characterized by comprising the following steps:
(1) Preparing recombinant protein p54-30-His and GST-p54-30;
(2) Preparing ASF positive control serum and ASF negative control serum;
(3) And (3) preparing a double-antigen sandwich ELISA kit for detecting ASFV antibodies.
Furthermore, the sequences of amplification primers used for detecting the recombinant protein of the ASFV antibody are shown in SEQ ID NO.3 and SEQ ID NO. 4.
The invention also provides application of the recombinant protein and the kit in preparation of a reagent for detecting the African swine fever virus antibody.
The preparation method of the double-antigen sandwich ELISA kit for detecting the ASFV antibody comprises the following steps:
(1) Recombinant protein production
artificially synthesizing ASFV p54 and p30 genes: optimizing codons of ASFV p54 and p30 genes to be suitable for being expressed in escherichia coli, respectively introducing restriction enzyme sites NdeI and XhoI at two ends of the genes of the p54 and p30 genes, synthesizing the genes, wherein the sequences are shown as SEQ ID NO.1, and cloning the synthesized genes into a vector pUC57;
b. constructing a recombinant bacterium: respectively carrying out restriction enzyme digestion on ASFV p54 and p30 genes in pET30a (+) plasmid and pUC57 by using restriction enzymes NdeI and XhoI, recovering the pET30a (+) plasmid after the restriction enzyme digestion and ASFV p54 and p30 genes of 726bp, connecting the recovered fragments by using T4 DNA ligase, transforming BL21 (DE 3) escherichia coli competent cells by using the connecting products, and screening out recombinant bacteria carrying plasmid pET-5430 by adopting a NdeI and XhoI double-enzyme cutting method; plasmid pET-5430 is used as template and primer
‘F 5’-CGCggatccCTATTCTCTTCAAGAAAG-3’(SEQ ID NO.3)，
R5 'GCAActcgagTTTTTTTCAGCAGTTTG-3' (SEQ ID NO. 4) PCR is amplified for ASFV P54 and P30 genes, the PCR product and pGEX-6P-1 plasmid are digested by restriction enzymes BamHI and XhoI respectively, the digested pGEX-6P-1 plasmid and ASFV P54 and P30 genes of 726bp are recovered by a nucleic acid recovery kit, the recovered fragments are ligated by T4 DNA ligase, the ligated product is transformed into BL21 (DE 3) Escherichia coli competent cells, and recombinant bacteria carrying the plasmid pGEX-5430 are screened by BamHI and XhoI double enzyme method.
Expressing and purifying ASFV p54 and p30 recombinant fusion protein: inoculating recombinant bacteria carrying plasmids pET-5430 and pGEX-5430 at a volume ratio of 1: 100 to 2 XYT medium containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin, respectively, and culturing at 37 ℃ to OD 600 =0.8, adding 1mmol/L IPTG, and inducing at 37 ℃ for 4h; centrifuging at 8000rpm for 2min, and centrifuging to obtain thallus and treating with 0.01M PWashing with BS solution for 2 times, and centrifuging again; the thalli obtained by centrifuging again is cracked by an ultrasonic instrument with the power of 50W,10 s/time for 10min; centrifuging at 12000rpm for 20min at 4 deg.C, and collecting precipitate; purifying recombinant proteins p54-30-His and GST-Binding Resin by respectively adopting Ni-NTA Agarose and GST-Binding Resin; taking the purified recombinant fusion protein, carrying out 2 mu g/channel and 12% polyacrylamide gel electrophoresis, transferring the protein to a nitrocellulose membrane by using a protein transfer printing instrument, and carrying out Western-blotting verification by using monoclonal antibodies against ASFV p30 and ASFV p 54.
(2) Preparation of control serum:
asf positive control serum: adjusting the concentration of the recombinant protein p54-30-His to 1mg/mL by using PBS (pH7.4 and 0.01M), mixing the recombinant protein p54-30-His with an equal volume of Freund's complete adjuvant, fully emulsifying by using a cell lysis instrument, and performing intramuscular injection of the emulsified recombinant protein into 45-day-old healthy pigs by 2 mL/pig; on the 10 th day after the first immunization, the antigen with the same dose mixed and emulsified by Freund incomplete adjuvant is used for boosting immunization for 1 time; after 10 days, the recombinant protein with the same dose is used for strengthening immunization for 1 time; on day 7 after the third immunization, serum was aseptically collected from swine blood; according to a conventional indirect ELISA method, 2.5 mu g/mL recombinant protein p54-30-His is respectively used for coating an ELISA plate, the ELISA plate is closed and then reacts with the serum separated from the pig which is continuously diluted by 10 times, the secondary antibody is an HRP-labeled goat anti-pig IgG (H + L) antibody which is diluted to a working concentration, the substrate is a TMB substrate solution, and the stop solution is 2M H 2 SO 4 Detecting the light absorption value of each hole under the wavelength of 450nm to determine that the ELISA antibody titer of the recombinant fusion protein immune pig serum is 1: 10 6 The serum is subpackaged and stored at-20 ℃ to be used as ASF positive control serum for detection.
Asf negative control serum: collecting blood of healthy pig of 75 days old, separating serum, subpackaging, and storing at-20 deg.C as ASF negative control serum for detection.
(3) Preparation of a double-antigen sandwich ELISA kit of an ASFV antibody:
a. 2 enzyme label plates: diluting the recombinant protein p54-30-His with a carbonate buffer solution with the pH value of 9.6 and 0.05M to the final concentration of 1 mu g/mL, adding 100 mu L of the buffer solution into each hole of a 96-hole plate, standing for 2h at the temperature of 37 ℃, washing for 2 times by using a washing solution PBST with the pH value of 7.4, adding 200 mu L of a sealing solution into each hole, standing for 1h at the temperature of 37 ℃, washing for 2 times by using the washing solution PBST, beating residual water on absorbent paper, air-drying in an ultra-clean workbench, filling into an aluminum foil bag, vacuumizing and sealing, and storing at the temperature of 4 ℃. Wherein the washing solution PBST component is 0.01M PBS, 0.5% Tween-20,0.01% Proclin300; the blocking solution is pH7.6, and contains 5% calf serum washing solution PBST.
b. Diluting liquid: 30mL of 5% calf serum containing 0.01% Proclin300, the specific preparation method is: completely dissolving 5mL of calf serum and 10 μ L of Proclin300 in PBST, diluting to 100mL of final volume, filtering and sterilizing by a 0.22 μm filter, taking 30mL of solution, subpackaging in a sterilization bottle, and storing at 4 ℃.
c. Reaction solution: the recombinant protein GST-p54-30 was diluted to 1. Mu.g/mL with a diluent, sterilized by filtration through a 0.22 μm filter, and then stored at 4 ℃ in a 25mL sterile vial.
d. Enzyme-labeled antibody: 10 mu L of mouse source anti-GST monoclonal antibody marked by HRP is preserved at the temperature of minus 20 ℃;
ASF positive control serum, ASF negative control serum: 1mL each, and stored at-20 ℃.
f.10 × washing solution: 30mL of 10-fold concentrated PBST, and specifically comprises the following steps: naCl 80g, KCl 2g, na 2 HPO 4 14.4g，KH 2 PO 4 2.4g, tween-20.5 mL, proclin 300.1 mL, dissolving in 800mL distilled water, adjusting pH to 7.4, metering to 1L, filtering and sterilizing by a 0.22 μm filter, taking 30mL, filling into a sterilization bottle, and storing at 4 ℃.
Tmb substrate solution: the TMB substrate solution was 25mL and stored at 4 ℃.
h. Stopping liquid: 2M H 2 SO 4 15mL, and storing at room temperature.
Advantageous effects
In the early experimental process, the recombinant antigen carrying the His tag is found to have the His tag interference phenomenon (His antibodies exist in the pig body immunized by the virus-like particle vaccine for many times) when the serum of the sow is detected. Recently, p30 can form a dimer in an experimental process, so that a carboxyl terminal part with high antigen index is only used for a p30 amino terminal sequence, a dimer forming region is deleted, the occurrence of false positive in the process of detecting an antibody by double-antigen sandwich ELISA is effectively avoided, a hydrophobic signal peptide region is removed from a used p54 antigen, and the expression quantity of a recombinant protein can be effectively improved; ASFV p54 and p30 fusion recombinant proteins which have excellent antigenicity and respectively carry His labels and GST labels are expressed and purified by a genetic engineering method, an ELISA plate is coated with the recombinant proteins of the His labels, serum to be detected is added, ASFV antibodies in the serum are combined on the ELISA plate, the ASFV recombinant proteins carrying GST labels are added, the proteins are captured by antibodies combined on the ELISA plate, an anti-GST monoclonal antibody marked by HRP is combined with the indirectly combined GST labels, and the existence of the ASFV antibodies is determined after the substrate solution is developed. The method can detect IgG antibodies and IgM antibodies generated in the early stage of an organism, and most importantly, eliminates the interference of common fusion tag protein, does not need to dilute a large amount of serum to be detected, has low background, and has important significance for accurately detecting ASFV antibodies.
Drawings
FIG. 1 is a diagram of a western blot of recombinant ASFV p54, p30 fusion proteins carrying different tags; wherein M is a Prestained Protein Marker; 1. 2 are purified recombinant proteins GST-p54-30, p54-30-His and anti-ASFV p30 monoclonal antibody immunoblotting results respectively; 3. 4 are purified recombinant proteins GST-p54-30, p54-30-His and a healthy mouse serum immunoblotting result respectively; 5. 6 are the result of immunoblotting of purified recombinant proteins GST-p54-30, p54-30-His and anti-ASFV p54 monoclonal antibody, respectively.
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 accompanying drawings and detailed description.
The source of the biological material is as follows:
pET30a (+): e.coli expression vectors, available from Novogen, USA (Cat No. 69909.3);
BL21 (DE 3) escherichia coli (e.coli) competent cells: purchased from sea-based organisms (HaiGene) technologies, ltd (Cat No. k 10127);
ASFV antibody positive serum: 12 ASFV antibody positive serum is provided by the national African swine fever reference laboratory of the China center for animal health and epidemiology;
ASFV antibody negative serum: 444 parts of ASFV antibody negative serum obtained in 2017, porcine Parvovirus (PPV) positive serum, porcine Foot and Mouth Disease Virus (FMDV) positive serum, porcine Reproductive and Respiratory Syndrome Virus (PRRSV) positive serum, porcine circovirus type 2 (PCV 2) positive serum and porcine pseudorabies virus (PRV) positive serum are identified and stored by the laboratory.
Enzyme-labeled antibody: the mouse-derived anti-GST monoclonal antibody labeled with HRP was purchased from ATAGENIXLABORATORIES (Cat No. ATMA10044Mo), the goat anti-pig IgG (H + L) antibody labeled with HRP was purchased from Abcam (Cat No. ab6915), and the goat anti-mouse IgG (H + L) antibody labeled with HRP was purchased from proteintech (Cat No. S00001).
Example 1 recombinant protein preparation
Artificial synthesis of ASFV p54, p30 gene: ASFV p54 and p30 gene codons are optimized to be suitable for being expressed in escherichia coli, restriction enzyme sites NdeI and XhoI are respectively introduced at two ends of the p54 and p30 genes, the genes are synthesized and inserted into a vector pUC57, and the gene sequences are as follows:
<xnotran> catatgCTATTCTCTTCAAGAAAGAAAAAAGCTGCTGCCGCTATTGAGGAGGAAGATATACAGTTTATAAATCCTTATCAAGATCAGCAATGGGCAGAAGTCACTCCACAACCAGGTACCTCTAAACCGGCTGGAGCGACTACAGCAAGTGCAGGCAAACCAGTCACGGGCAGACCGGCAACAAACAGACCAGCAACAAACAAACCAGTCACGGACAACCCAGTTACGGACAGACTAGTCATGGCAACTGGCGGGCCAGCGGCCGCACCTGCGGCCGCGAGTGCTCATCCGACTGAGCCTTACACGACAGTCACTACTCAGAACACTGCTTCACAAACAATGTCGGCTATTGAAAATTTACGACAAAGAAACACCTATACGCATAAAGACCTAGAAAACTCCTTGGATATCGAAGAAGAAACCGAATCTTCTGCTTCTTCTGAATCTATCCACGAAAAAAACGACAACGAAACCAACGAATGCACCTCTTCTTTCGAAACCCTGTTCGAACAGGAACCGTCTTCTGAAGAACCGAAAGACTCTAAACTGTACATGCTGGCTCAGAAAACCGTTCAGCACATCGAACAGTACGGTAAAGCTCCGGACTTCAACAAAGTTATCCGTGCTCACAACTTCATCCAGACCATCCACGGTACCCCGCTGAAAGAAGAAGAAAAAGAAGTTGTTCGTCTGATGGTTATCAAACTGCTGAAAAAAAAActcgag (SEQ ID NO. 1), ASFV LFSSRKKKAAAAIEEEDIQFINPYQDQQWAEVTPQPGTSKPAGATTASAGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAHPTEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSLDIEEETESSASSESIHEKNDNETNECTSSFETLFEQEPSSEEPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIHGTPLKEEEKEVVRLMVIKLLKKK (SEQ ID NO. 2), aa1-aa133 p54 , aa134-aa 238 p30 . </xnotran>
2. Constructing a recombinant bacterium: the method comprises the steps of cutting the ASFV p54 and p30 genes in pET30a (+) plasmid and pUC57 by restriction enzymes NdeI and XhoI respectively, recovering the cut pET30a (+) plasmid and ASFV p54 and p30 genes with 726bp, connecting the recovered fragments by T4 DNA ligase, transforming BL21 (DE 3) escherichia coli competent cells by the connecting products, screening recombinant plasmids by double enzyme cutting of NdeI and XhoI, and naming as pET-5430; plasmid pET-5430 is used as template and primer
F 5’-CGCggatccCTATTCTCTTCAAGAAAG-3(SEQ ID NO.3)，
R5.
3, ASFV p54, p30 recombinant fusion protein expression and purification: inoculating recombinant bacteria carrying plasmids pET-5430 and pGEX-5430 to 2 XYT culture medium containing 50. Mu.g/mL kanamycin and 100. Mu.g/mL ampicillin at a volume ratio of 1: 100, respectively, and culturing at 37 ℃ to OD 600 =0.8, add 1mmol/L IPTG, induce 4h at 37 ℃; 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 power of 50W,10 s/time, for 10min; centrifuging at 4 deg.C and 12000rpm for 20min, and collecting precipitate; respectively adopting Ni-NTA Agarose and GST Binding Resin to purify recombinant protein (p 54-30-His) carrying His label and recombinant protein (GST-p 54-30) carrying GST label; taking the purified recombinant fusion protein 2 mu g/channel, carrying out electrophoresis separation on 12% polyacrylamide gel, transferring a nitrocellulose membrane by using a protein transfer printing instrument, and carrying out Western-blotting verification by using monoclonal antibodies against ASFV p30 and ASFV p54 (figure 1).
EXAMPLE 2 preparation of Positive control serum and negative control
Asf positive control serum: adjusting the concentration of the purified p54-30-His recombinant protein to 1mg/mL by using PBS (pH7.4 and 0.01M), mixing with an equal volume of Freund's complete adjuvant, and fully emulsifying in a tissue homogenizer; injecting the water-in-oil recombinant fusion protein into 45-day-old healthy pigs by intramuscular injection, wherein each pig is 2 mL; 10 days after the first immunization, 1 booster immunization with the same dose of antigen mixed with Freund's incomplete adjuvant; after 10 days, the recombinant fusion protein with the same dosage is used for strengthening immunity for 1 time; on day 7 after the third immunization, swine blood serum was aseptically collected; according to a conventional indirect ELISA method, 2.5 mu g/mL recombinant protein p54-30-His is respectively used for coating an ELISA plate, the ELISA plate is closed and then reacts with the continuous 10-fold diluted pig separation serum, a secondary antibody is an HRP-labeled goat anti-pig IgG antibody diluted to a working concentration, a substrate is a TMB substrate solution, and a stop solution is 2M H 2 SO 4 Detecting the light absorption value of each hole under the wavelength of 450nm to determine that the ELISA antibody titer of the recombinant fusion protein immune pig serum is 1: 10 6 The serum is subpackaged and stored at-20 ℃ to be used as ASF positive control serum for detection.
Asf negative control serum: collecting blood of healthy pig of 75 days old, separating serum, subpackaging, and storing at-20 deg.C as ASF negative control serum for detection.
Example 3 establishment of double antigen sandwich ELISA method for ASFV antibody
Optimization of ELISA reaction conditions: the recombinant protein p54-30-His is coated on an enzyme label plate with different concentrations, and is sealed by different types of sealing liquid (including 5% skim milk, 2% BSA, 5% calf serum, 5% gelatin and the like), p54-30 antiserum and negative control serum with different dilution times, GST-p54-30 reaction liquid with different concentrations are subjected to combined reaction, and the reaction time is optimized, so that the final determination: the optimal effect is that the recombinant protein p54-30-His is diluted by 0.05M carbonate buffer solution (pH9.6) to 8 mu g/mL and coated on an ELISA plate at 37 ℃ for 2h, the calf serum with the concentration of 5% is sealed at 37 ℃ for 45min, the serum is diluted 5 times and reacts at 37 ℃ for 45min, GST-p54-30 with the concentration of 1 mu g/mL reacts for 45min, and TMB substrate solution reacts for 15 min.
Determination of ELISA cut-off value and experimental establishment conditions: 200 parts of ASFV negative serum are detected according to the optimized reaction conditions, and the wave length is 450nmLong lower reading of absorbance (OD) of each well sample 450 ) And calculating the average value of the readings
And standard deviation SD, to->
For the cut-off value, the cut-off value of the ELISA method was finally determined to be 0.091. Repeating ASFV positive control serum and ASFV negative control serum 10 times respectively by the above method, and calculating the average value of each reading>
And standard deviation, in->
Value sum>
The value was used as a conditional cut-off value for establishment of positive and negative control sera, and the result showed OD of the positive control serum 450 Need to be more than or equal to 1.510, negative control serum OD 450 Less than or equal to 0.087.
Example 4 preparation of double antigen sandwich ELISA kit for ASFV antibody
1. Antibody detection enzyme label plate: the reagent box contains 2 processed enzyme labeling plates with 96 holes (packaged by an aluminum foil bag), and the enzyme labeling plate processing process is as follows: the recombinant protein p54-30-His was diluted with 0.05M carbonate buffer (pH9.6) to a final concentration of 1. Mu.g/mL, 100. Mu.L was added to each well of a 96-well plate, incubated at 37 ℃ for 2 hours, washed 2 times with washing solutions PBST (0.01MPBS, 0.5% Tween-20, 0.01-procilin 300, pH7.4), then blocked with blocking solutions (0.01M PBS,5% calf serum, 0.01-procilin 300, pH7.6) at 37 ℃ for 1 hour, washed 3 times with PBST, blotted on absorbent paper, air-dried in an ultraclean bench, the enzyme-labeled plate and a desiccant were packed in an aluminum foil bag (1 plate/bag), vacuum-sealed and stored at 4 ℃.
2. Diluting liquid: 30mL of 5% calf serum containing 0.01% (w/w) Proclin300, and the preparation method comprises: completely dissolving 5mL of calf serum and 10 mu of LProclin300 in PBST, fixing the volume to 100mL finally, filtering and sterilizing by a 0.22 mu m filter, taking 30mL, filling into a sterilization bottle, and storing at 4 ℃;
3. reaction solution: diluting the recombinant protein GST-p54-30 to 1 mu g/mL by using a diluent, filtering and sterilizing by using a 0.22 mu m filter, filling into a 25mL sterilization bottle, and storing at 4 ℃;
4. enzyme-labeled antibody: 10 mu L of mouse source anti-GST monoclonal antibody marked by HRP is preserved at the temperature of minus 20 ℃;
ASFV positive control serum, ASF negative control serum: each 1mL of the solution is stored at-20 ℃;
6.10 × washing solution: 30mL of 10-fold concentrated PBST, and the preparation method specifically comprises the following steps: naCl 80g, KCl 2g, na 2 HPO 4 14.4g，KH 2 PO 4 2.4g, tween-20.5 mL, proclin 300.1mL, dissolving in 800mL of distilled water, adjusting pH to 7.4, fixing the volume to 1L, filtering and sterilizing by a 0.22-micron filter, taking 30mL, filling into a sterilization bottle, and storing at 4 ℃;
tmb substrate solution: TMB substrate solution 25mL,4 degrees C storage;
8. stopping liquid: 2M H 2 SO 4 15mL, and storing at room temperature.
And (4) aseptically packaging the components in bottles and assembling to obtain the double-antigen sandwich ELISA kit.
Example 5 use of a Dual antigen Sandwich ELISA kit for ASFV antibodies
1. The microplate was removed from the kit and allowed to equilibrate to room temperature (16-26 deg.C) to avoid direct sunlight.
2. Diluting the serum to be detected and ASFV antibody positive and negative control serum by a diluent with the dilution multiple of 5 times, respectively adding the diluted sample into corresponding holes of an ELISA plate at a concentration of 100 mu L/hole, and acting at 37 ℃ for 45min;
washing the ELISA plate with 3.1 times of washing solution, washing the ELISA plate with 200 mu L/hole for 3 times and 3 min/time, and patting the liquid in the hole on absorbent paper;
4. adding 100 μ L/well of reaction solution, and reacting at 37 deg.C for 45min;
5. adding enzyme-labeled antibody diluted by 1: 10000, acting for 45min at 37 ℃ in 100 mu L/hole;
washing the enzyme label plate with 6.1 times of washing liquid, and drying liquid in the holes on absorbent paper after washing for 3 min/time and 200 mu L/hole times of 3 times;
7. adding TMB substrate solution at a concentration of 100 μ L/hole, and standing in dark at room temperature for 15min;
8. adding stop solution to stop the reaction, and stopping the reaction at 50 mu L/hole;
9. reading the light absorption value (OD 450) of 450nm wavelength of each hole by using a microplate reader;
10. OD in control well corresponding to ASFV positive serum 450 Not less than 1.510, OD of ASFV positive serum control hole 450 Under the precondition of less than or equal to 0.087, the OD of the serum sample to be detected 450 More than 0.091, the ASFV antibody is judged to be positive; OD 450 Less than or equal to 0.091, and the ASFV antibody is judged to be negative.
Example 6 double antigen Sandwich ELISA kit specificity assay for ASFV antibody
The prepared ASFV antibody double-antigen sandwich ELISA kit is used for detecting the positive serum of common porcine viral diseases, and the kit comprises Porcine Parvovirus (PPV) positive serum, porcine Foot and Mouth Disease Virus (FMDV) positive serum, porcine Reproductive and Respiratory Syndrome Virus (PRRSV) positive serum, porcine circovirus type 2 (PCV 2) positive serum and porcine pseudorabies virus (PRV) positive serum, and the kit is determined whether to have cross reaction with the serum, and the result shows that the sample detection is negative (Table 1).
TABLE 1 kit specificity test results
Example 7 clinical application of double-antigen sandwich ELISA kit for ASFV antibody
456 clinical serum samples are detected by the ASFV antibody ELISA kit, wherein 12 ASFV antibody positive sera and 444 ASFV antibody negative sera are detected by the kit. The result shows that the detection results of 12 ASFV antibody positive sera are all positive, and the antibody positive detection accuracy rate is 100%; in 444 parts of ASFV antibody negative serum, 425 parts of detection results are negative, and the antibody negative detection accuracy rate is 95.7%; the overall detection compliance rate is 95.8%.
Example 8 in-batch and inter-batch reproducibility test of double-antigen sandwich ELISA kit for ASFV antibody
1. In-batch repeatability test: random3 enzyme label plates in the same batch are extracted to be balanced for 30min at room temperature, 20 parts of ASFV antibody negative pig serum and 9 parts of ASFV antibody positive serum are detected according to the established double-antigen sandwich ELISA method, and the detection is carried out according to OD 450 The value was calculated as the Coefficient of Variation (CV) within the batch, which showed a CV of 7.18% or less (Table 2).
TABLE 2 results of repeated tests in batches of ELISA test kit for ASFV antibody
2. Batch to batch repeatability test: randomly extracting 1 enzyme label plate of different batches, balancing at room temperature for 30min, detecting 20 parts of ASFV antibody negative pig serum and 9 parts of ASFV antibody positive serum according to the established double-antigen sandwich ELISA method, and detecting according to OD 450 The inter-batch coefficient of variation was calculated and the result showed a CV of 6.56% or less (Table 3).
TABLE 3 results of repeated tests among batches of ELISA test kit for ASFV antibody
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.
Claims (10)
1. The recombinant protein for detecting the ASFV antibody is characterized in that the recombinant protein takes ASFV p54 and p30 as antigens, the sequence of the recombinant protein is shown as SEQ ID No.1, and the amino acid sequence of the ASFV antigen is shown as SEQ ID No. 2.
2. The method of claim 1, wherein restriction enzyme sites NdeI and XhoI are introduced into both ends of the p54 and p30 genes, respectively, and the resulting product is inserted into a vector pUC 57.
3.A double-antigen sandwich ELISA kit for detecting ASFV antibody, characterized in that it comprises the recombinant protein for detecting ASFV antibody, ELISA plate, HRP-labeled anti-ELISA-labeled antibody, ASF positive control serum, ASF negative control serum, diluent, reaction solution, 10 Xwashing solution, TMB substrate solution and stop solution of claim 1.
4. The double-antigen sandwich ELISA kit for detection of ASFV antibody according to claim 3 wherein the antigen coating 96-well ELISA plate in the kit is His tag carrying recombinant ASFV p54, p30 recombinant protein p54-30-His; the reaction solution is recombinant ASFV p54 and p30 recombinant protein GST-p54-30 carrying GST tags.
5. The double-antigen sandwich ELISA kit for detecting ASFV antibody according to claim 4, wherein the recombinant protein p54-30-His is obtained by expression and purification in BL21 (DE 3) E.coli using the plasmid pET-30a (+) loaded with the recombinant protein according to claim 1; the recombinant protein GST-P54-30 is obtained by expressing and purifying plasmid pGEX-6P-1 loaded with the recombinant protein of claim 1 in BL21 (DE 3) Escherichia coli.
6. The kit of claim 3, wherein the HRP-labeled anti-GST enzyme-labeled antibody is an HRP-labeled mouse anti-GST monoclonal antibody; the ASF positive control serum is prepared from recombinant p54 and p30 fusion protein immune pigs; the ASF negative control serum is healthy pig serum.
7. The double-antigen sandwich ELISA kit for detection of ASFV antibody according to claim 3,
the diluent is 5% calf serum containing 0.01% proclin300;
the 10 × washing solution is: naCl 80g, KCl 2g, na 2 HPO 4 14.4g，KH 2 PO 4 2.4g, tween-20.5 mL, proclin 300.1mL, dissolving in 800mL of distilled water, adjusting pH to 7.4, fixing the volume to 1L, filtering and sterilizing by a 0.22 mu m filter, and storing at 4 ℃;
the stop solution is 2 MH 2 SO 4 。
8. The method for preparing a double-antigen sandwich ELISA kit for detection of ASFV antibody according to any of claims 3 to 7, characterized by comprising the steps of:
preparing recombinant protein p54-30-His and GST-p54-30;
preparing ASF positive control serum and ASF negative control serum;
and (3) preparing a double-antigen sandwich ELISA kit for detecting ASFV antibodies.
9. The method for preparing a double-antigen sandwich ELISA kit for detecting an ASFV antibody according to claim 8, wherein the amplification primer sequences for detecting the recombinant protein of the ASFV antibody are shown in SEQ ID No.3 and SEQ ID No. 4.
10. Use of the recombinant protein according to claim 1 and the kit according to claim 3 for the preparation of a reagent for the detection of antibodies to African swine fever virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211064046.0A CN115947795B (en) | 2022-09-01 | 2022-09-01 | Recombinant protein for detecting ASFV antibody, double-antigen sandwich ELISA kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211064046.0A CN115947795B (en) | 2022-09-01 | 2022-09-01 | Recombinant protein for detecting ASFV antibody, double-antigen sandwich ELISA kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115947795A true CN115947795A (en) | 2023-04-11 |
| CN115947795B CN115947795B (en) | 2024-04-16 |
Family
ID=87284722
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211064046.0A Active CN115947795B (en) | 2022-09-01 | 2022-09-01 | Recombinant protein for detecting ASFV antibody, double-antigen sandwich ELISA kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115947795B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103278627A (en) * | 2013-05-22 | 2013-09-04 | 扬州大学 | Multi-antigen ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting African swine fever virus antibody |
| CN104497136A (en) * | 2014-12-05 | 2015-04-08 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Monoclonal antibody for African swine fever virus gene II type strain as well as preparation method and application thereof |
| CN110684085A (en) * | 2019-11-07 | 2020-01-14 | 青岛立见诊断技术发展中心 | ASFV-P54 protein recombination method and sandwich ELISA kit prepared by using same |
| CN111393510A (en) * | 2020-04-02 | 2020-07-10 | 中国农业科学院兰州兽医研究所 | African swine fever virus recombinant antigen and application thereof |
| CN111929433A (en) * | 2019-08-30 | 2020-11-13 | 洛阳普泰生物技术有限公司 | African swine fever virus antibody ELISA detection kit and preparation method thereof |
| AU2020103187A4 (en) * | 2020-09-09 | 2021-01-14 | Chinese Academy Of Inspection And Quarantine | A Monoclonal Antibody Capable Of Competing With Positive Serum For Binding To African Swine Fever Virus B646L Antigen And The Application Thereof |
| EP3835425A1 (en) * | 2019-06-17 | 2021-06-16 | Bioapplications Inc. | Recombinant vector for producing antigen for diagnosis of african swine fever and use thereof |
| KR20210106181A (en) * | 2020-02-20 | 2021-08-30 | 한국생명공학연구원 | Antigen protein composition for diagnosing infection of African Swan Fever and use thereof |
| CN113698474A (en) * | 2020-05-21 | 2021-11-26 | 河南省生物工程技术研究中心 | African swine fever polyclonal antibody and African swine fever antigen detection test strip |
| CN114152748A (en) * | 2021-10-29 | 2022-03-08 | 浙江理工大学绍兴生物医药研究院有限公司 | Double-antibody sandwich ELISA diagnostic kit for detecting African swine fever virus and method thereof |
| US20220193218A1 (en) * | 2020-12-21 | 2022-06-23 | VST LLC dba Medgene Labs | African swine fever (asf) virus vaccines |
| US20220249648A1 (en) * | 2019-08-02 | 2022-08-11 | Ming-Che Shih | Recombinant baculovirus displaying african swine fever virus proteins, and an immunological composition comprising the same |
-
2022
- 2022-09-01 CN CN202211064046.0A patent/CN115947795B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103278627A (en) * | 2013-05-22 | 2013-09-04 | 扬州大学 | Multi-antigen ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting African swine fever virus antibody |
| CN104497136A (en) * | 2014-12-05 | 2015-04-08 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Monoclonal antibody for African swine fever virus gene II type strain as well as preparation method and application thereof |
| EP3835425A1 (en) * | 2019-06-17 | 2021-06-16 | Bioapplications Inc. | Recombinant vector for producing antigen for diagnosis of african swine fever and use thereof |
| US20220249648A1 (en) * | 2019-08-02 | 2022-08-11 | Ming-Che Shih | Recombinant baculovirus displaying african swine fever virus proteins, and an immunological composition comprising the same |
| CN111929433A (en) * | 2019-08-30 | 2020-11-13 | 洛阳普泰生物技术有限公司 | African swine fever virus antibody ELISA detection kit and preparation method thereof |
| CN110684085A (en) * | 2019-11-07 | 2020-01-14 | 青岛立见诊断技术发展中心 | ASFV-P54 protein recombination method and sandwich ELISA kit prepared by using same |
| KR20210106181A (en) * | 2020-02-20 | 2021-08-30 | 한국생명공학연구원 | Antigen protein composition for diagnosing infection of African Swan Fever and use thereof |
| CN111393510A (en) * | 2020-04-02 | 2020-07-10 | 中国农业科学院兰州兽医研究所 | African swine fever virus recombinant antigen and application thereof |
| CN113698474A (en) * | 2020-05-21 | 2021-11-26 | 河南省生物工程技术研究中心 | African swine fever polyclonal antibody and African swine fever antigen detection test strip |
| AU2020103187A4 (en) * | 2020-09-09 | 2021-01-14 | Chinese Academy Of Inspection And Quarantine | A Monoclonal Antibody Capable Of Competing With Positive Serum For Binding To African Swine Fever Virus B646L Antigen And The Application Thereof |
| US20220193218A1 (en) * | 2020-12-21 | 2022-06-23 | VST LLC dba Medgene Labs | African swine fever (asf) virus vaccines |
| CN114152748A (en) * | 2021-10-29 | 2022-03-08 | 浙江理工大学绍兴生物医药研究院有限公司 | Double-antibody sandwich ELISA diagnostic kit for detecting African swine fever virus and method thereof |
Non-Patent Citations (8)
| Title |
|---|
| GENBANK: "AAY46108.1: structural protein p54, partial [African swine fever virus]", 《GENBANK》, 26 July 2016 (2016-07-26) * |
| GUANGLEI ZHANG等: "Antigenicity and immunogenicity of recombinant proteins comprising African swine fever virus proteins p30 and p54 fused to a cell-penetrating peptide", 《INTERNATIONAL IMMUNOPHARMACOLOGY》, vol. 101, 29 October 2021 (2021-10-29), pages 108251, XP086887656, DOI: 10.1016/j.intimp.2021.108251 * |
| UMA BHARATHI INDRABALAN等: "An extensive evaluation of codon usage pattern and bias of structural proteins p30, p54 and, p72 of the African swine fever virus (ASFV)", 《VIRUSDISEASE》, vol. 32, no. 04, pages 810 - 822, XP037630736, DOI: 10.1007/s13337-021-00719-x * |
| YUNLEI CAO等: "Identification of one novel epitope targeting p54 protein of African swine fever virus using monoclonal antibody and development of a capable ELISA", 《RESEARCH IN VETERINARY SCIENCE》, vol. 141, 7 October 2021 (2021-10-07), pages 19 - 25, XP093032669, DOI: 10.1016/j.rvsc.2021.10.008 * |
| 冯春燕等: "非洲猪瘟病毒p54抗原C端的原核表达及多克隆抗体的制备与鉴定", 《动物医学进展》, vol. 40, no. 06, 22 October 2018 (2018-10-22), pages 1 - 6 * |
| 姜睿姣等: "非洲猪瘟检测技术进展", 《病毒学报》, vol. 35, no. 03, 27 May 2019 (2019-05-27), pages 523 - 532 * |
| 张丽: "基于p54蛋白单克隆抗体的非洲猪瘟病毒抗原检测方法的建立", 《中国优秀硕士学位论文全文数据库》, no. 2021, 15 September 2021 (2021-09-15) * |
| 李杰等: "非洲猪瘟病毒p30-54融合蛋白基因的构建、表达及其多克隆抗体制备", 《河南农业科学》, vol. 47, no. 09, pages 126 - 130 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115947795B (en) | 2024-04-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103278627B (en) | A kind of multi-antigen ELISA kit detecting African swine fever virus antibody | |
| CN1609617B (en) | Composition and method for diagnosing and preventing serious acute respiratory syndrome(SARS) | |
| CN110698543A (en) | Double-antigen indirect ELISA kit for African swine fever virus antibody detection | |
| CN110873792B (en) | African swine fever virus antibody detection kit | |
| CN106432440B (en) | PPA-ELISA (polymerase chain reaction-enzyme linked immunosorbent assay) detection kit for Brucella melitensis antibody and preparation method of PPA-ELISA detection kit | |
| CN103059109B (en) | Mycoplasma pneumonia antigen, preparation method and immunodetection kit | |
| CN113607952B (en) | African swine fever virus blocking ELISA antibody detection kit and preparation method and application thereof | |
| CN103293306A (en) | Preparation method for African swine fever virus antibody detection colloidal gold immunochromatography test paper strip | |
| CN106442981B (en) | A kind of 1 type antibody indirect ELISA diagnostic kit of human bocavirus | |
| CN111732664A (en) | Novel coronavirus recombinant protein, rabbit-human chimeric antibody, preparation method and application thereof | |
| CN112505330A (en) | Novel coronavirus detection kit based on fusion protein of nucleocapsid protein | |
| CN115160411A (en) | Screening, preparation and application of African swine fever virus dominant antigen | |
| CN106518989B (en) | Polypeptide for detecting porcine Delta coronavirus antibody, preparation method and application thereof | |
| CN108776225A (en) | Pig parvoviral VLPs antibody assay kits and preparation method thereof, application | |
| CN105061602A (en) | Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof | |
| CN114736290A (en) | Nano antibody capable of identifying porcine pseudorabies virus with high accuracy and sensitivity, preparation method and application | |
| Cheng et al. | Development of a quick dot blot assay for the titering of bovine ephemeral fever virus | |
| CN111621506B (en) | Mycoplasma bovis secretory protein Mbovp0145 and application thereof | |
| US20230375548A1 (en) | Indirect enzyme-linked immunosorbent assay detection kit based on p30 protein and p22 protein of african swine fever virus | |
| CN116023506B (en) | ASFV nonstructural protein dominant antigen epitope fusion protein, kit and application thereof | |
| CN115947795B (en) | Recombinant protein for detecting ASFV antibody, double-antigen sandwich ELISA kit and application thereof | |
| CN109799342A (en) | A kind of O-shaped antibodies against foot-and-mouth disease virus competitive ELISA detection kit | |
| CN106279408B (en) | Monoclonal antibody and antibody combination for resisting foot-and-mouth disease type O virus and application of monoclonal antibody and antibody combination in detection of virus antigen and antibody | |
| CN110229218B (en) | Reagent for detecting Seneca virus antibody and polypeptide used by same | |
| CN110196325B (en) | In-plug virus diagnosis kit and test paper |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |