CN104497137A - General monoclonal antibody for African swine fever virus strains as well as preparation method and application thereof - Google Patents
General monoclonal antibody for African swine fever virus strains as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN104497137A CN104497137A CN201410736335.XA CN201410736335A CN104497137A CN 104497137 A CN104497137 A CN 104497137A CN 201410736335 A CN201410736335 A CN 201410736335A CN 104497137 A CN104497137 A CN 104497137A
- Authority
- CN
- China
- Prior art keywords
- swine fever
- african swine
- fever virus
- monoclonal antibody
- hybridoma
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/91—Cell lines ; Processes using cell lines
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a general monoclonal antibody for African swine fever virus strains as well as a preparation method and application thereof. The preparation method of the general monoclonal antibody for the African swine fever virus strains comprises the following steps: immunizing a mouse by adopting a purified p54 immune protein, fusing spleen cells of the immunized mouse with myeloma cells to prepare hybridoma cells, screening with three screening antigens so as to obtain the hybridoma cells which can stably secrete a monoclonal antibody, and then preparing the monoclonal antibody by adopting an in vivo or in vitro method, wherein the three screening antigens comprise a prokaryotically expressed p54 recombinant protein, an eukaryotically expressed p54 recombinant protein and an artificially synthesized polypeptide shown in Seq ID No.1. The preparation method of the general monoclonal antibody for the African swine fever virus strains has the advantages that the three screening antigens are adopted for screening the hybridoma cells, and especially an artificially synthesized common polypeptide of all the African swine fever virus strains is taken as the screening antigen, so that the prepared general monoclonal antibody can be used for detecting strains in different geographic regions, the misdetection rate caused by regional difference among the strains is reduced, and the quarantine working quality and efficiency are improved.
Description
Technical field
The application relates to monoclonal antibody art, particularly relates to a kind of general purpose single clonal antibody for African swine fever virus strain, and the preparation method and application of this monoclonal antibody.
Background technology
African swine fever (African Swine Fever, ASF) be by African swine fever virus (African Swine Fever Virus, the one of the pig ASFV) caused is acute, hot, high degree in contact communicable disease, the high heat of its clinical symptom performance, dermohemia, miscarriage, oedema and internal organs are hemorrhage, and lethality rate is up to 100%.But after man of Ye You minority virus strain infection pig, there is subacute infection or carriers in pig.OIE (OIE) is classified as category-A animal epidemic, be subject to the great attention of countries in the world, not yet there is this disease in China, this disease is defined as a class zoonosis by China, and as animal exotic carry out studying (Sun Huaichang. Chinese Preventive Veterinary Medicine report, 1999,21 (21): 117-119).
In recent years, African swine fever spans to the Armenia in the Eurasia, Ukraine, Russia by the African continent, and particularly Caucasus region epidemic situation is severe in recent years, causes great threat to China.African swine fever in West Europe, the generation in South America and Eastern Europe is verified, African swine fever is once import into and carry out destructive strike by industrial belt of raising pigs.African swine fever is listed in National Animal Disease monitor in 2011 plan by China, determines to attach most importance in Xinjiang, the Inner Mongol, Heilungkiang, Jilin, Liaoning, Tibet etc. and monitors provinces and regions (Wang Hongyan, raises pigs, 2011,4:81-82).
There is no effective vaccine at present, therefore the infection conditions of pig is judged by quick diagnosis African swine fever antibody horizontal, thus formulate effective control and the measure of elimination, and then diagnostic detection promptly and accurately and strict effective to block the measure such as to slaughter be the effective ways successfully eliminating African swine fever.African swine fever antibody diagnosis method comprises agar diffusion test, ELISA, immunoblotting, immunofluorescence etc.At present, the main dependence on import of detection reagent of China's diagnosis African swine fever, the method and the reagent that have independent intellectual property right are little, and the epidemic situation situation of surrounding countries' sternness in addition, China is in the urgent need to setting up oneself detection method and reagent.
Further, in existing antibody diagnosis method, prepare antigen owing to adopting live virus cells infected and to cause when clinical application trace routine cannot normalizing operation, and have a big risk because operating poison alive, be therefore not suitable for promoting the use.At present for the multiplex gene engineering method antigen expressed of exploitation of detection method and reagent, mainly concentrate on research as isogenic in p72, p54, p32, pp62, A104R, B602L, K205R, wherein the application such as p72, p54 is wider.And the preparation of monoclonal antibody also mainly concentrates on these structural protein, set up thus based in the current world wide of commercial reagents box of immunological method only four.But, because areal variation is large between African swine fever sick virus stain, makes existing commercial reagents box in use, easily occur the detection leakage phenomenon of the African swine fever disease caused by some strain.
Summary of the invention
The object of the application is to provide a kind of new African swine fever virus strain general purpose single clonal antibody, and the preparation method and application of this general purpose single clonal antibody.
To achieve these goals, the application have employed following technical scheme:
The application discloses a kind of preparation method of African swine fever virus strain general purpose single clonal antibody on the one hand, comprise and adopt the p54 immune protein of purifying to carry out immunity to mouse, get immune mouse spleen cell and murine myeloma cell to merge and prepare hybridoma, then three kinds of screening antigens are adopted to screen positive hybridoma cell strain, obtain Absorbable organic halogens secretion anti-African swine fever virus monoclonal antibody hybridoma, then by body or external method prepare African swine fever virus strain general purpose single clonal antibody; Wherein, three kinds of screening antigens comprise, prokaryotic expression p54 recombinant protein, eukaryotic expression p54 recombinant protein, and the polypeptide of sequence shown in the Seq ID No.1 of synthetic;
Seq ID No.1：SSRKKKAAAAIEEEDIQFINPYQDQQWAEV。
It should be noted that, the key of the application is that employing three kinds screening antigen screens hybridoma cell strain, the wherein polypeptide of sequence synthetic shown in Seq ID No.1, specially designed for existing all African swine fever virus strains, this sequence through comparing in ncbi database, for all African swine fever virus strains are common, therefore, monoclonal antibody prepared by the hybridoma cell strain filtered out by it possesses versatility, can detect the African swine fever virus in different geographical source, thus avoid undetected problem.
Also it should be noted that, the key of the application is that employing three kinds screening antigen screens hybridoma cell strain, especially creationaryly devises an artificial synthetic polypeptide, thus makes the monoclonal antibody prepared possess versatility; Merge the actual conditions of hybridoma and step as the preparation of p54 immune protein and purifying, the preparation of prokaryotic expression p54 recombinant protein, the preparation of eukaryotic expression p54 recombinant protein, preparation, the immunoscreening etc. of hybridoma can carry out with reference to existing ordinary method, tiredly to state at this.After screening obtains the hybridoma of Absorbable organic halogens secretion anti-African swine fever virus monoclonal antibody, in the body of monoclonal antibody or external preparation method, also can with reference to existing ordinary method; Such as, by hybridoma abdominal injection animal, as mouse, gather ascites, separation and purification monoclonal antibody; Or, the monoclonal antibody of secretion generation is collected by this hybridoma of vitro culture.
Through the hybridoma of the Absorbable organic halogens secretion anti-African swine fever virus monoclonal antibody that screening obtains, called after hybridoma cell strain SZCIQASFV1, its microbial preservation number is: CCTCC No.C2014212; Classification And Nomenclature is: hybridoma hybridoma cell; The preservation time is: on December 3rd, 2014; Depositary institution is: China typical culture collection center; Preservation address is: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center.
In the application, p54 immune protein obtains in the following manner, p54 gene fragment in pMD18-T-p54 plasmid is proceeded to pET-52b (+) plasmid, and carry out solubility expression in bacillus coli DH 5 alpha, then extraction purification expressing protein, i.e. p54 immune protein.
In the application, prokaryotic expression p54 recombinant protein is for utilizing pET-52b (+) plasmid p54 recombinant protein of extraction purification after prokaryotic expression in intestinal bacteria; Eukaryotic expression p54 recombinant protein is the p54 recombinant protein utilizing pFastBac/NT-TOPO insect expression system eukaryotic expression to obtain.
The another side of the application discloses a kind of African swine fever virus strain general purpose single clonal antibody, and this general purpose single clonal antibody is secreted by the hybridoma cell strain SZCIQASFV1 of preserving number CCTCC No.C2014212 and produced.
It should be noted that, in fact, the African swine fever virus strain general purpose single clonal antibody of the application prepares acquisition by the preparation method of the application.
The one side again of the application discloses the application of general purpose single clonal antibody in preparation African swine fever virus detection reagent or equipment of the application.
The one side again of the application specifically discloses a kind of African swine fever virus enzyme-linked immunologic detecting kit of the general purpose single clonal antibody containing the application.
On the basis of above research, present invention also provides a kind of hybridoma secreting the African swine fever virus strain general purpose single clonal antibody of the application, its preserving number is CCTCC No.C2014212.
It should be noted that, the hybridoma of the secretion African swine fever virus strain general purpose single clonal antibody of the application, in fact, be exactly African swine fever virus strain general purpose single clonal antibody preparation method in screen acquisition Absorbable organic halogens secretion anti-African swine fever virus monoclonal antibody hybridoma.
Owing to adopting above technical scheme, the beneficial effect of the application is:
The preparation method of the application, adopt three kinds to screen antigen to screen hybridoma, particularly design the total polypeptide of synthetic all African swine fever virus strain are as screening antigen, the monoclonal antibody prepared can be detected the African swine fever virus strain in different geographical source, substantially reduce the detection leakage phenomenon that areal variation between strain causes, improve inspection and quarantine work quality and efficiency.
Accompanying drawing explanation
Fig. 1: the PCR qualification result being recombinant expression plasmid pET-52b (+) 3C/LIC-p54 in the embodiment of the present application;
Fig. 2: the SDS-PAGE analytical results being the abduction delivering product of pET-52b (+) 3C/LIC-p54 recombinant plasmid in BL21 (DE3) in the embodiment of the present application;
Fig. 3: the purifying and the results of immunoblot analysis that are the abduction delivering product of pET-52b (+) 3C/LIC-p54 recombinant plasmid in BL21 (DE3) in the embodiment of the present application;
Fig. 4: the PCR qualification result being pFastBac-p54 recombinant plasmid and Bacmid-p54 recombinant cosmid in the embodiment of the present application;
Fig. 5: the Western-blot qualification result being pFastBac/NT-TOPO insect expression system eukaryotic expression p54 recombinant protein in the embodiment of the present application;
Fig. 6: be the result figure adopting ProteinOn XPR36 macromole to make the affinity of specific antigen epitope polypeptide A in instrument analysis African swine fever virus strain general purpose single clonal antibody and African swine fever virus p54 albumen mutually in the embodiment of the present application;
Fig. 7: the compare of analysis result figure being African swine fever virus p54 protein sequence in the embodiment of the present application.
Embodiment
Because areal variation between African swine fever virus strain is large, makes existing commercial reagents box in use, easily occur detection leakage phenomenon, need a kind of monoclonal antibody can all the sick virus stain of the African swine fever in different geographical source with good detection effect badly, therefore, the application is intended to develop a strain monoclonal antibody, this monoclonal antibody can with all known African swine fever virus strain effect of current world-wide prevalence, thus provide critical material and method for the immunological method such as competitive ELISA method set up based on monoclonal antibody, solve the problem that existing commercial reagents box is undetected, and minimizing maybe need not rely on external detection reagent, China is made to have autonomous detectivity and detection reagent, be strictly on guard against that African swine fever epidemic situation imports China into.
Based on above demand and object, the application designs and the common polypeptide of synthetic all known African swine fever virus strain especially, the i.e. polypeptide of sequence shown in Seq ID No.1, for filtering hybridoma, simultaneously, adopt prokaryotic expression p54 recombinant protein and eukaryotic expression p54 recombinant protein to screen, the monoclonal antibody of final preparation can be detected existing all known African swine fever virus strains, i.e. African swine fever virus strain general purpose single clonal antibody.Wherein, the polypeptide as screening antigen of synthetic, confirm through ncbi database comparison, in theory, the monoclonal antibody of especially screening acquisition can detect all known African swine fever virus strains; Also test the strain in different geographical source in the test of the application, result conforms to theory.
It should be noted that, in the application, shown in the Seq ID No.1 of synthetic, the polypeptide of sequence designs for antigen site " EDIQFINPYQ ", in order to ensure immune effect, so be designed to sequences polypeptide shown in Seq ID No.1; Be appreciated that, on the basic conception of the application, when ensureing that antigen site " EDIQFINPYQ " is complete, can on the basis of sequences polypeptide shown in Seq ID No.1, before and after peptide sequence, increase and decrease about 20 amino acid, all belong to the protection domain of the application; When not considering synthetic cost, more amino acid can also be increased on sequences polypeptide basis shown in the Seq ID No.1 of the application.
On the basis of above research, this application provides African swine fever virus strain general purpose single clonal antibody, and application.Be appreciated that the monoclonal antibody of the application has good versatility, can to different geographical source strain detect, substantially reduce because of strain areal variation cause undetected; Therefore, test kit can be prepared into and be widely used in inspection and quarantine work; This test kit, except containing except the monoclonal antibody of the application, also contains the conventional reagent of other enzyme linked immunosorbent detection certainly, does not tire out state at this.
Below by specific embodiments and the drawings, the application is described in further detail.Following examples are only further described the application, should not be construed as the restriction to the application.
The preparation of embodiment 1 immunizing antigen
1. the amplification of goal gene and purifying
Design pair of primers, has the pMD18-T-p54 plasmid of African swine fever virus p54 gene from clone the target gene that increases; Wherein pMD18-T-p54 plasmid is preserved by Animal &. Plant Inspection and Quarantine Techn Center, Shenzhen Bureau of Impor provides, containing, for example the p54 gene of the 552bp of sequence shown in Seq ID No.10 in this plasmid.The upstream primer of this primer is sequence shown in Seq ID No.2, and downstream primer is sequence shown in Seq ID No.3.
Seq ID No.2：5’-CAGGGACCCGGTTATACTATTCTCATTGCTATCG-3’
Seq ID No.3：5’-GGCACCAGAGCGTTCAAGGAGTTTTCTAGGTC-3’
After primer Synesis Company synthesizes above primer, with pMD18-T-p54 plasmid for template carries out pcr amplification, reaction conditions is 94 DEG C of 1min, 58 DEG C of 1min, 30cycles, 72 DEG C of 5min.
Reaction terminates the agarose gel electrophoresis pcr amplification product of rear employing 1.5%, and blend compounds reclaims test kit and reclaims purifying target DNA, namely obtains target gene.
2. the structure of recombinant plasmid
In the target gene that above-mentioned steps obtains, add T4DNA polysaccharase, hatch 30min for 22 DEG C, hatch 20min for 75 DEG C, add 3C/LIC carrier, hatch 5min for 22 DEG C, after adding EDTA, hatch 5min for 22 DEG C, build pET-52b (+) 3C/LIC-p54 recombinant plasmid.
3. the conversion of recombinant plasmid and qualification
PET-52b (+) the 3C/LIC-p54 recombinant plasmid that above-mentioned steps obtains is proceeded to bacillus coli DH 5 alpha, and be inoculated on the LB nutrient agar medium containing 100mg/L penbritin, cultivate 12h for 37 DEG C, the single bacterium colony of picking in containing the LB liquid nutrient medium of 50mg/L penbritin 37 DEG C cultivate after 12h, extract plasmid, carry out PCR qualification and order-checking, detect the integrity of target gene.
Wherein PCR qualification have employed two pairs of primers respectively and carries out, and first to primer pair shown in the Seq ID No.2 of the target gene that namely increases and Seq ID No.3, and second pair of primer is the T7 primer pair that pET-52b (+) 3C/LIC plasmid carries.As shown in Figure 1, in figure, M swimming lane is DNAmarker to the Gel electrophoresis results of PCR qualification, and 1 swimming lane is the electrophoresis result of first pair of primer PCR amplification, and 2 swimming lanes are the electrophoresis result of second pair of primer PCR amplification; Visible, pET-52b (+) the 3C/LIC-p54 construction of recombinant plasmid success of this example, containing the target gene of the 462bp that has an appointment, conforms to theory expectation.Containing the p54 gene of 462bp in sequencing result display recombinant plasmid, shown in its sequence with Seq ID No.10, sequence conforms to, and pET-52b (+) the 3C/LIC-p54 recombinant plasmid demonstrating this example structure further contains the p54 gene required for us.
It should be noted that, the front end of p54 gene comprises two promotors, and during this routine prokaryotic expression, only cloned one of them promotor, namely the signal peptide nucleotide sequence of the front end 90bp size of p54 gene is cloned, so first is 462bp to primer pair amplifies product size shown in the Seq ID No.2 of the target gene that namely increases and Seq ID No.3, this gene contains the full gene sequence of p54 albumen equally, does not just comprise the signal peptide nucleotide sequence of front end 90bp; Concerning this example, do not affect the prokaryotic expression of p54 albumen.
4. the abduction delivering of recombinant plasmid in BL21 (DE3)
Preparation BL21 (DE3) bacterium competent cell, after recombinant plasmid pET-52b (+) 3C/LIC-p54 being transformed into BL21 (DE3) cell, cultivating containing on the LB flat board of 100mg/L penbritin, the single bacterium colony of picking, be inoculated in 2mL containing in the LB nutrient solution of 100mg/L penbritin, 37 DEG C, 200r/min constant-temperature table shaking culture to OD600 value is about 0.6.Add the IPTG that final concentration is 2mM, after cultivating 2h, collect bacterium liquid.By nutrient solution at 4 DEG C of centrifugal 15min, remove supernatant, collect thalline.By the resuspended precipitation of PBS, 4 DEG C of centrifugal 15min of 10000g, remove supernatant.With the resuspended thalline of broken bacterium damping fluid, ultrasonication on ice, 4 DEG C of centrifugal 15min, collect supernatant.Coomassie brilliant blue staining is carried out to expression product, observations with sex change discontinuous SDS-PAGE.
As shown in Figure 2, in figure, M swimming lane is protein molecular quality standard to result; 1-5 swimming lane is respectively 0.5mM, and 1mM, 2mM, 4mM, 8mM IPTG induces the analytical results of pET-52b (+) 3C/LIC-p54 bacteria lysis supernatant after two hours; 6 swimming lanes are do not add the analytical results that IPTG induces pET-52b (+) 3C/LIC-p54 bacteria lysis supernatant after two hours; 7 swimming lanes are the analytical results that 1mMIPTG induces BL21 (DE3) bacteria lysis supernatant after two hours; 8-12 swimming lane is respectively 1mM IPTG and induces pET-52b (+) 3C/LIC-p541h, the analytical results of bacteria lysis supernatant after 2h, 3h, 4h, 5h; 13 swimming lanes are do not add the analytical results that IPTG induces pET-52b (+) 3C/LIC-p54 bacteria lysis supernatant after two hours; 14 swimming lanes are that 1mMIPTG induces BL21 (DE3) bacteria lysis supernatant after two hours.Result shows, the p54 recombinant protein needed for expression that pET-52b (+) the 3C/LIC-p54 recombinant plasmid that this example builds can be stable.
5. the qualification of expression product
By the bacterium liquid that above-mentioned steps is cultivated, at 4 DEG C of centrifugal 15min, remove supernatant, collect thalline.By the resuspended precipitation of PBS, 4 DEG C of centrifugal 15min of 10000g, remove supernatant.With the resuspended thalline of broken bacterium damping fluid, ultrasonication on ice, 4 DEG C of centrifugal 15min, collect supernatant.African swine fever standard positive serum is adopted to carry out immunoblotting assay to expression product, with the antigenicity of testing goal albumen.As shown in Figure 3, in figure, M is protein standard to results of immunoblot analysis, and 1 is sample washing lotion, protein purification sample for the purpose of 2, and 3 is sample dissociation liquid, Western blot analysis for the purpose of 4; Result shows, and the recombinant protein that this example obtains is p54 recombinant protein really.
6. the purifying of expression product
Adopt the supernatant liquor of Ni post affinity chromatography method purifying ultrasonication on ice, the immunizing antigen of purified product namely required for this example, i.e. p54 immune protein.
Embodiment 2 screens the preparation of antigen
This example have employed three kinds of screening antigens and screens hybridoma, and one is the prokaryotic expression p54 recombinant protein as immunizing antigen in embodiment 1, using the p54 immune protein after ni-sepharose purification as screening antigen; Two be utilize the method for synthetic to synthesize Seq ID No.1 shown in polypeptide; Three is utilize pFastBac/NT-TOPO insect expression system eukaryotic expression p54 recombinant protein, will infect recombinant baculovirus cell ultrasonication supernatant as screening antigen.
To be this example propose polypeptide shown in the Seq ID No.1 of wherein synthetic on the basis than the right p54 protein sequence of disclosed all African swine fever viruses at present, the peptide sequence that all African swine fever viruses are total, therefore, it is as screening antigen, can filter out the general purpose single clonal antibody of all African swine fever viruses in theory, comparing result as shown in Figure 7.
Being prepared as follows of antigen:
Seq ID No.1：SSRKKKAAAAIEEEDIQFINPYQDQQWAEV
1. the synthesis of primer
3 groups of primers are synthesized according to pFastBac/NT-TOPO insect expression system, first group is p54 gene amplification primer pair, upstream primer NT-TOPO-ASFV54F is sequence shown in Seq ID No.4, and downstream primer NT-TOPO-ASFV54R is sequence shown in Seq ID No.5; Second group is primer pair pFastBac/NT-TOPO-p54 recombinant plasmid being carried out to PCR qualification, its upstream primer Polyhedrin forward primer is sequence shown in Seq ID No.6, and downstream primer SV40polyA reverse primer is sequence shown in Seq ID No.7; 3rd group is primer pair Bacmid-p54 recombinant cosmid being carried out to PCR qualification, and its upstream primer pUC/M13Forward is sequence shown in Seq ID No.8, and downstream primer pUC/M13Reverse is sequence shown in Seq ID No.9.
Seq ID No.4：5’-ATGGATTCTGAATTTTTTCAACC-3’
Seq ID No.5：5’-TTACAAGGAGTTTTCTAGGTCT-3’
Seq ID No.6：5’-AAATGATAAC CATCT CGC-3’
Seq ID No.7：5’-GGTAT GGCTGATTAT GATC-3’
Seq ID No.8：5’-CCCAG TCACGACGTT GTAAAACG-3’
Seq ID No.9：5’-AGCGGATAAC AATTT CACAC AGG-3’
The structure of 2.pFastBac-p54 restructuring transposon vector
Using the pMD18T-p54 recombinant plasmid identical with embodiment 1 as pcr template, with first group of primer, namely p54 gene amplification primer increases to p54 gene, reclaims PCR primer, namely obtain target gene with PureLink HiPure Mini Plasmid Purification Kit.Get 4 μ L target genes to be connected with 1 μ L pFastBac/NT-TOPO carrier, then transform DH5 α competent cell.
Picking positive colony bacterium, adopts above-mentioned two groups of primers to carry out PCR qualification: one is adopt primer pair NT-TOPO-ASFV54F and NT-TOPO-ASFV54R of amplification target gene to carry out PCR, i.e. first group of primer.Two is adopt primer pair NT-TOPO-ASFV54F and SV40polyAreverse primer.Result shows, and first group of primer amplification obtains the fragment of about 552bp, conforms to the p54 gene of the 552bp of expection; Second group of primer amplification obtains clip size and is about 758bp, and illustration purpose gene has been connected to carrier and has been applicable to position, and closure is correct, obtains pFastBac-p54 recombinant plasmid.The partial gel electrophoresis result of pcr amplification is as shown in the A figure of Fig. 4, and the electrophoresis result that wherein, M is DNAMarker, swimming lane 1 and 2 is all second group of primer extension product, 1 is that negative bacterium colony plasmid amplification product, 2 is for positive bacterium colony plasmid amplification product.
3. recombinant cosmid Bacmid-p54 builds
By 10 μ L recombinant plasmid pFastBac-p54 Transformed E .coli DH 10Bac competent cells.Transformed and afterwards in EP pipe, added 900 μ L LB nutrient solutions, 37 DEG C of shaking table 225rpm cultivate 4h.With room temperature, LB nutrient solution presses 1:10,1:100 and 1:1000 tri-kinds of concentration dilution bacterium liquid, the LB be coated on respectively containing 50 μ g/mL kantlex (hereinafter referred to as K+), 7 μ g/mL gentamicins (hereinafter referred to as G+), 10 μ g/mL tsiklomitsins (hereinafter referred to as Tet+), the X-Gal of 100 μ g/mL and the IPTG of 40 μ g/mL screens on flat board, is inverted for 37 DEG C and cultivates 48h.Picking at least 10 hickies, are inoculated on the screening flat board of above-mentioned composition, line point single bacterium colony, are inverted overnight incubation for 37 DEG C, are confirmed whether to be still hickie.Finally be defined as the bacterium colony renewed vaccination of hickie in the LB nutrient solution of 20mL, nutrient solution contains K+, G+ and Tet+, 37 DEG C of shaking table 225rpm overnight incubation.Bacmid-CAT and Bacmid-HTA is done same operation simultaneously, be set to the positive and negative control respectively.Take out 1 part of bacterium liquid, adopt Large plasmid to extract test kit PureLink HiPure Plasmid DNAMiniprep Kit and extract Bacmid, all the other bacterium liquid 4 DEG C preservation.PCR qualification is carried out: one is employing first group of primer, and primer pair NT-TOPO-ASFV54F and NT-TOPO-ASFV54R of the target gene that namely increases carries out PCR with two groups of primers; Two is employing the 3rd group of primers, and namely primer pair pUC/M13Forward and pUC/M13Reverse carries out PCR.The partial gel electrophorogram of pcr amplification product is as shown in the B figure of Fig. 4, and wherein the amplified production, 4 of M to be DNA Marker, 3 be first group of primer and primer pair NT-TOPO-ASFV54F and NT-TOPO-ASFV54R is the amplified production, 5 of the 3rd group of primer and primer pair pUC/M13Forward and pUC/M13Reverse is negative control.Result shows, and first group of primer amplification obtains the fragment of about 552bp, conforms to the p54 gene of the 552bp of expection; 3rd group of primer amplification obtains the fragment that size is about 2988bp, illustrates that p54 gene has been connected on baculovirus vector.After having identified, the remaining bacterium solution preparation that 4 DEG C are preserved is become glycerol stock and frozen at-80 DEG C.
4. the acquisition of recombinant baculovirus AcNPV-p54
Press
iI Reagent transfection reagent box operation instructions, Bacmid-p54, Bacmid-CAT, Bacmid-HTA is lipidization.Lipidization rear transfection Sf 9 insect cell, simultaneously not having the normal Sf9 insect cell of transfection Bacmid DNA to be set to blank.Cell is put 28 DEG C of constant incubators and cultivates about 72h, period notes observation of cell pathology situation.There is pathology in about 36 hours later cell, collecting cell culture supernatant appears pathology, in 72 hours 80% cells, wherein containing recombinant baculovirus.Supernatant infects normal cell again, observation of cell pathology situation, and pathology appears in 72 hours 80% cells, cleer and peaceful cell in collection.To the cell ultrasonication of recombinant baculovirus be infected, extract supernatant as screening antigen.
Western-blot qualification is carried out to screening antigen, shown in result figure Fig. 5, in figure, M is protein marker, 1 is normal sf9 cell protein, 2 for p54 recombinant protein; Result shows, the eukaryotic expression p54 recombinant protein containing expection in supernatant liquor
The preparation of embodiment 3 monoclonal antibody
1. animal immune
The purifying p54 immune protein of preparation in embodiment 1 is mixed and emulsification with the Freund's complete adjuvant of equivalent, the female mouse of subcutaneous injection BALB/c in 7 week age, every only 0.2 μ g.Mix with p54 albumen and emulsification with equivalent Freund's incomplete adjuvant after 14d, subcutaneous multi-point injection.Mix with p54 albumen and emulsification with equivalent Freund's incomplete adjuvant after 21d, subcutaneous multi-point injection.Before plan and SP2/0 cytogamy, 3d does not add the booster immunization injection of any adjuvant.
2. the foundation of positive hybridoma cell strain
Get high immune mouse of tiring, mixed by its splenocyte with SP2/0 myeloma cell in 10: 1 ratios, conventional PEG fusion method prepares hybridoma.Adopt the screening antigen of preparation in embodiment 2 to carry out indirect ELISA method and detect every hole Hybridoma Cell Culture supernatant, and establish intestinal bacteria, Sf9 cell protein as the negative control screening antigen.
Get the hole of the three kinds of screening antigen indirect ELISA result strong positives simultaneously met in embodiment 2, limiting dilution assay is adopted to carry out subclone, the hybridoma dilution of continuous 5 times and the monoclonal antibody test of continuous 5 times, and last 3 antibody tests, total positives rate hole reaches 100%, obtains the hybridoma cell strain of stably excreting monoclonal antibody.
3. the preparation of monoclonal antibody
Get the female mouse of BALB/c in 10 week age, abdominal injection whiteruss, every 0.5mL, 1 week backward abdominal injection, 5 × 106 positive hybridoma cells.After 7-10d, mouse web portion obviously swells, and collect ascites with the centrifugal 5min of 2000r/min, supernatant liquor is the ascites containing anti-African swine fever p54 protein monoclonal antibody, i.e. the African swine fever virus strain general purpose single clonal antibody of this example ,-80 DEG C save backup.
4. the qualification of monoclonal antibody subclass
Return to room temperature by with the reagent in the monoclonal antibody subgroup identification test kit of Sigma company, in the small test tube that test card is housed, first add 500 μ L damping fluids, then add the mouse ascites of 500 μ L collections, rock small test tube gently and make the abundant submergence of test card.Add 1mL anti-mouse antibody colloidal gold binding substances again, keep test card to have the one side of word to soak all the time in a liquid, rock small test tube in room temperature with gentle, until there is point.Through qualification, the monoclonal antibody of this strain of hybridoma secretion is IgG1 subclass, conforms to expection.
5. monoclonal antibody specificity qualification
Adopt respectively Pestivirus suis, PRRS virus, foot and mouth disease virus, swine influenza virus, porcine pseudorabies virus totivirus albumen bag by elisa plate, as detectable antigens, the African swine fever virus strain general purpose single clonal antibody adopting this example to prepare is as tested antibody, detect through indirect ELISA method, result shows African swine fever virus strain general purpose single clonal antibody and the equal no cross reaction of above-mentioned Prevention of Common Occurrence Porcine Disease cause of disease of the preparation of this example, has good specificity.
6. monoclonal antibody affinity is analyzed
Utilize ProteinOnXPR36 macromolecule interaction system, study the affinity of specific antigen epitope polypeptide A in the African swine fever virus strain general purpose single clonal antibody of this example preparation and African swine fever virus p54 albumen, judge between antigen-antibody in conjunction with dissociation degree, thus judge the stability of immune complex.First whether activate chip, be combined on chip by African swine fever virus strain general purpose single clonal antibody prepared by this example by chemical bonded refractory, then being deactivated by chip and testing monoclonal antibody combines firm.Using the polypeptide A of different concns for preparing as liquid phase, with 25 μ L/min flow velocitys by combining the solid phase chip of monoclonal antibody, again by antigen antibody complex dissociation after the combination of antigen-antibody reaches peak value, test its dissociation rate.
As shown in Figure 6, in figure, X-coordinate 0s-150s is that different concns polypeptide A and be combineding with each other of monoclonal antibody act on to result, and 150s-500s is dissociating of immune complex; The polypeptide A, 6 of the polypeptide A, 5 of the polypeptide A, 4 of the polypeptide A, 3 of 1 is concentration be the polypeptide A, 2 of 150nM to be concentration be 75nM to be concentration be 37.5nM to be concentration be 18.75nM to be concentration be 9.375nM is PBST damping fluid.
Result shows, this example preparation African swine fever virus strain general purpose single clonal antibody and African swine fever virus p54 albumen in the dissociation rate constant of specific antigen epitope polypeptide A when room temperature (Kd) be 6.13 × 10-41/s, its equilibrium association constant (KD) is 2.97 × 10-9M, and association rate constant (Ka) is 2.06 × 1051/Ms; Show that the combination of African swine fever virus strain general purpose single clonal antibody prepared by this example and specific antigen epitope polypeptide A is better, avidity is comparatively strong, is suitable for checkout and diagnosis.
The application of embodiment 4 monoclonal antibody
1. materials and methods
1.1 serum and c-ELISA test kit
African swine fever standard positive serum is purchased from Britain IAH Pirbright laboratory; 153 parts of negative serums are collected in domestic multiple pig farm; C-ELISA commercial kit is purchased from INGENASA company of Spain.
The preparation of 1.2 envelope antigens
Adopt purifying antigen prepared by embodiment 1, i.e. p54 immune protein, measure protein concentration, for bag by elisa plate.
The preparation of 1.3 monoclonal antibodies
Adopt African swine fever virus strain general purpose single clonal antibody prepared by embodiment 3, and the upper horseradish peroxidase (HRP) of mark ,-80 DEG C save backup.
1.4 competitive ELISA schedule of operation
Bag quilt: P54 immune protein antigen coating buffer is diluted to 2.5 μ g/mL, joins in 96 hole enzyme plates respectively, and every hole 100 μ L, puts 4 DEG C of refrigerator overnight, or 37 DEG C of effect 1h.
Wash plate: take out enzyme plate and discard liquid, every hole adds washing lotion 200 μ L, washes plate, totally three times.Last inversion pats dry.
Close: every hole adds confining liquid 100 μ L, 37 DEG C of reaction 1h, wash plate.
Application of sample: sample and weak positive control, positive control, negative control diluent do 1:5 dilution, and mixing, adds ELISA Sptting plate, every hole 50 μ L respectively; 37 DEG C of reaction 1h, wash plate.
Add the enzyme mark monoclonal antibody binding substances in 1.3: every hole adds 50 μ L IgG-HRP, mixes gently.Enzyme plate puts 37 DEG C of effect 30min, and reaction completes, and washes plate.
Add substrate solution: every hole adds 100 μ L substrate solutions, 37 DEG C of effect 10min.
Termination reaction: take out Sptting plate, every hole adds 50 μ L stop buffers fast, surveys its OD value in 30min.
Survey light absorption value: by microplate reader under 450nm wavelength, measure its light absorption value.
The optimization of 1.5 competitive ELISA methods experiment conditions
1.5.1 antigen coated concentration screening
By indirect ELISA experimental procedures, adopt matrix arrangement, respectively the p54 after purifying is pressed concentration gradient bag quilt: 5,2.5,1.25,0.625 μ g/mL, 50 μ L/ holes, wrap at 4 DEG C and spent the night.Enzyme mark monoclonal antibody 06-HRP vertical setting of types doubling dilution: 25 ×-3200 ×, 0.5h at 37 DEG C, colour developing 3min, adds 0.5M sulfuric acid 50 μ L/ hole color development stopping, chooses about OD1.5, and considers bag by cost and the number using enzyme mark monoclonal antibody, selects best bag by concentration.
1.5.2 enzyme mark monoclonal antibody working concentration screening
By above-mentioned 1.4 competitive ELISA experimental procedures, adopt the p54 purifying protein coated elisa plate of 2.5 μ g/mL, standard positive serum to be checked respectively by 40 ×, 80 ×, 160 ×, 320 × longitudinal doubling dilution, and do repetition; Standard female serum to be checked compares by 40 × dilution, also does repeating hole.Laterally add competition 06-HRP monoclonal antibody, and press gradient dilution: 100 ×, 200 ×, 400 ×, 800 ×.Measure OD value, calculate inhibiting rate, get the best effort concentration of the highest monoclonal antibody extension rate of inhibiting rate as enzyme mark monoclonal antibody.
1.6 competitive ELISA methods and results decision thresholds are determined
For determining that competitive ELISA test method judges the threshold value of yin and yang attribute result, this experiment employing 20 parts of negative serums are as tested serum, these 20 parts of serum are detected as negative serum through the African swine fever antibody ELISA detection kit of INGENASA company of Spain, the competitive ELISA method test program good by above-mentioned optimization is tested, and then measures the OD value of every part of serum.Calculate the blocking-up rate of these 20 parts of serum according to the following formula, i.e. PI value:
PI (%)=(1-Sample serum mean OD value ÷ negative serum control mean OD value) × 100%
Calculate mean P I value and its standard deviation S of 20 parts of serum.Add that the value of 3 times of standard deviation S judges threshold value as positive findings by mean P I value; Add that the value of 2 times of standard deviation S judges threshold value as negative findings by mean P I value; Be positioned at and be then judged to be suspicious specimen between the two.
1.7 competitive ELISA methods are applied to and detect field sample
Fetch the 107 parts of serum samples coming from country variant, positive serum samples and 54 parts of negative serum samples of different strain are infected comprising 53 parts, the working specification good by above-mentioned optimization and criterion detect, and carry out accordance with commercial detection test kit and compare.
2 results
The screening of 2.1 envelope antigen concentration
By indirect ELISA experimental procedures, adopt matrix arrangement, respectively the p54 after purifying is pressed concentration gradient bag quilt: 5,2.5,1.25,0.625 μ g/mL, 50 μ L/ holes, wrap at 4 DEG C and spent the night.Above-mentioned enzyme mark monoclonal antibody vertical setting of types doubling dilution: 25 ×-3200 ×, 0.5h at 37 DEG C, colour developing 3min, adds 0.5M sulfuric acid 50 μ L/ hole color development stopping.Result is as shown in table 1.
Table 1 antigen concentration the selection result
Choose OD450nm place light absorption value about 1.5 albumen bag by concentration and monoclonal antibody concentration, with reference to monoclonal antibody titration result, consider that the less test combinations of antigen-antibody consumption is as best operating condition, determines that albumen the best bag is 2.5 μ g/mL by concentration simultaneously.
The screening of 2.2 best enzyme mark monoclonal antibody working concentrations
By above-mentioned 1.4 competitive ELISA experimental procedures, adopt the p54 purifying protein coated elisa plate of 2.5 μ g/mL, standard positive serum to be checked respectively by 40 ×, 80 ×, 160 ×, 320 × longitudinal doubling dilution, and do repetition; Standard female serum to be checked compares by 40 × dilution, also does repeating hole.Laterally add above-mentioned competition enzyme mark monoclonal antibody, and press gradient dilution: 100 ×, 200 ×, 400 ×, 800 ×.Measure OD value, shown in result table 2.
Table 2 best enzyme mark monoclonal antibody working concentration the selection result
Calculate inhibiting rate, get the best effort concentration of the highest monoclonal antibody extension rate of inhibiting rate as enzyme mark monoclonal antibody.When enzyme mark monoclonal antibody dilutes 100 times, the inhibiting rate of positive serum is the highest, therefore the extension rate of enzyme mark monoclonal antibody selects 100 times as best enzyme mark monoclonal antibody working concentration.
The determination of 2.3 competitive ELISA method threshold values and criterion
This experiment employing 20 parts of local negative serums are as tested serum, and these 20 parts of serum are negative serum through business detection kit, and the competitive ELISA method test program good by above-mentioned optimization is tested, and then measures the OD value of every part of serum.Calculate mean P I value and its standard deviation S of 20 parts of serum.Add that the value of 3 times of standard deviation S judges threshold value as positive findings by mean P I value; Add that the value of 2 times of standard deviation S judges threshold value as negative findings by mean P I value; Be positioned at and be then judged to be suspicious specimen between the two, concrete outcome is as shown in table 3.
Table 3 threshold value calculation result
2.4 competitive ELISA method field tests
Fetch the 107 parts of serum samples coming from country variant, positive serum samples and 54 parts of negative serum samples of different strain are infected comprising 53 parts, the working specification good by above-mentioned optimization and criterion detect, and carry out accordance with commercial detection test kit and compare.Result shows, and 53 parts of positive serum samples that the method that this experiment is set up detects are all positive, and 54 parts of negative serum samples are all negative.And detect 51 parts in the 53 parts of positive serum samples adopting the African swine fever antibody ELISA detection kit of INGENASA company of Spain to detect.
Visible, the competitive ELISA method that the African swine fever virus strain general purpose single clonal antibody adopting this example to prepare is set up effectively can detect the African swine fever virus strain that different geographical is originated, and recall rate is 100%, not undetected.The African swine fever virus strain general purpose single clonal antibody of this example and competitive ELISA method thereof, can effective substituting import one test kit, sets up and meets the detection reagent of international trade and the animal epidemic monitoring method of applicable China's national situation.
Above content is the further description done the application in conjunction with concrete embodiment, can not assert that the concrete enforcement of the application is confined to these explanations.For the application person of an ordinary skill in the technical field, under the prerequisite not departing from the application's design, some simple deduction or replace can also be made, all should be considered as the protection domain belonging to the application.
Claims (9)
1. the preparation method of an African swine fever virus strain general purpose single clonal antibody, it is characterized in that: comprise and adopt the p54 immune protein of purifying to carry out immunity to mouse, get immune mouse spleen cell and murine myeloma cell to merge and prepare hybridoma, then three kinds of screening antigens are adopted to screen positive hybridoma cell strain, obtain Absorbable organic halogens secretion anti-African swine fever virus monoclonal antibody hybridoma, then by body or external method prepare African swine fever virus strain general purpose single clonal antibody; Described three kinds of screening antigens comprise, the polypeptide of sequence shown in the Seq ID No.1 of prokaryotic expression p54 recombinant protein, eukaryotic expression p54 recombinant protein and synthetic;
Seq ID No.1：SSRKKKAAAAIEEEDIQFINPYQDQQWAEV。
2. preparation method according to claim 1, it is characterized in that: described p54 immune protein obtains in the following manner, p54 gene fragment in pMD18-T-p54 plasmid is proceeded to pET-52b (+) plasmid, and solubility expression is carried out in bacillus coli DH 5 alpha, then extraction purification expressing protein, i.e. p54 immune protein.
3. preparation method according to claim 1, is characterized in that: described prokaryotic expression p54 recombinant protein is for utilizing pET-52b (+) plasmid p54 recombinant protein of extraction purification after prokaryotic expression in intestinal bacteria; Described eukaryotic expression p54 recombinant protein is the p54 recombinant protein utilizing pFastBac/NT-TOPO insect expression system eukaryotic expression to obtain.
4. an African swine fever virus strain general purpose single clonal antibody, is characterized in that: described general purpose single clonal antibody is secreted by the hybridoma cell strain SZCIQASFV1 of preserving number CCTCC No.C2014212 and produced.
5. general purpose single clonal antibody according to claim 4, is characterized in that: described general purpose single clonal antibody is prepared by the method described in any one of claim 1-3.
6. the application of the general purpose single clonal antibody according to claim 4 or 5 in preparation African swine fever virus detection reagent or equipment.
7. an African swine fever virus enzyme-linked immunologic detecting kit, is characterized in that: containing the general purpose single clonal antibody described in claim 4 or 5 in described test kit.
8. secrete a hybridoma for African swine fever virus strain general purpose single clonal antibody, its preserving number is CCTCC No.C2014212.
9. the preparation method of hybridoma according to claim 8, comprise and adopt the p54 immune protein of purifying to carry out immunity to mouse, get immune mouse spleen cell and murine myeloma cell to merge and prepare hybridoma, then three kinds of screening antigens are adopted to screen positive hybridoma cell strain, limiting dilution assay cell subclone is carried out to the sample that three kinds of screening antigens are strong positive, the hybridoma dilution of continuous several times and the monoclonal antibody test of continuous several times, and last three antibody test total positives rate holes reach the hybridoma of 100%, namely the hybridoma of Absorbable organic halogens secretion anti-African swine fever virus monoclonal antibody is obtained,
Described three kinds of screening antigens comprise, the polypeptide of sequence shown in the Seq ID No.1 of prokaryotic expression p54 recombinant protein, eukaryotic expression p54 recombinant protein and synthetic;
Seq ID No.1：SSRKKKAAAAIEEEDIQFINPYQDQQWAEV。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410736335.XA CN104497137B (en) | 2014-12-05 | 2014-12-05 | The general monoclonal antibody of African swine fever virus strain and preparation method and application |
| PCT/CN2015/075183 WO2016086554A1 (en) | 2014-12-05 | 2015-03-27 | General monoclonal antibody for african swine fever virus strains as well as preparation method therefor and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410736335.XA CN104497137B (en) | 2014-12-05 | 2014-12-05 | The general monoclonal antibody of African swine fever virus strain and preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104497137A true CN104497137A (en) | 2015-04-08 |
| CN104497137B CN104497137B (en) | 2017-10-31 |
Family
ID=52938604
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410736335.XA Expired - Fee Related CN104497137B (en) | 2014-12-05 | 2014-12-05 | The general monoclonal antibody of African swine fever virus strain and preparation method and application |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN104497137B (en) |
| WO (1) | WO2016086554A1 (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109734810A (en) * | 2019-01-24 | 2019-05-10 | 深圳市雅臣智能生物工程有限公司 | Anti- African swine fever virus and CD dual-target pig source antibody, the preparation method and application |
| CN109810948A (en) * | 2019-01-18 | 2019-05-28 | 中国农业科学院兰州兽医研究所 | The hybridoma cell strain of anti-African swine fever virus K205R protein monoclonal antibody and its antibody of secretion |
| CN111549001A (en) * | 2020-05-28 | 2020-08-18 | 嘉铭(固安)生物科技有限公司 | Hybridoma cell strain secreting African swine fever virus p34 protein monoclonal antibody, monoclonal antibody and application |
| CN111849922A (en) * | 2020-07-20 | 2020-10-30 | 华中农业大学 | Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof |
| CN111925436A (en) * | 2019-11-29 | 2020-11-13 | 洛阳普泰生物技术有限公司 | Monoclonal antibody of African swine fever virus P30 protein and application thereof |
| CN112481220A (en) * | 2020-11-03 | 2021-03-12 | 中国农业科学院兰州兽医研究所 | anti-African swine fever virus helicase D1133L monoclonal antibody, hybridoma cell strain secreting monoclonal antibody and application |
| CN112661817A (en) * | 2020-12-25 | 2021-04-16 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Preparation method, epitope identification and application of African swine fever virus p54 protein monoclonal antibody |
| CN113045673A (en) * | 2021-03-25 | 2021-06-29 | 深圳海关动植物检验检疫技术中心 | African swine fever virus antibody detection kit based on chimeric P54 epitope and application |
| CN113402601A (en) * | 2021-06-09 | 2021-09-17 | 河南中泽生物工程有限公司 | Preparation method and application of anti-African swine fever virus p54 protein monoclonal antibody |
| CN114167055A (en) * | 2021-10-21 | 2022-03-11 | 山东绿都生物科技有限公司 | Competitive enzyme-linked immunosorbent assay kit for detecting anti-African swine fever antibody in serum |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937349B (en) * | 2017-11-27 | 2021-01-15 | 中国检验检疫科学研究院 | Cell line for stably expressing African swine fever virus P54 protein and preparation and application thereof |
| CN109517072A (en) * | 2018-11-20 | 2019-03-26 | 杭州贤至生物科技有限公司 | A kind of preparation of typhoid bacillus recombinant protein and its monoclonal antibody |
| CN109735567A (en) * | 2019-01-18 | 2019-05-10 | 扬州大学 | A kind of the recombinant adenoviral vector building and adenovirus packing method of African swine fever EP153R and P54 gene co-expressing |
| CN110423761B (en) * | 2019-07-08 | 2022-07-05 | 郑州大学 | African swine fever virus antibody detection test paper |
| CN112444626B (en) * | 2019-08-30 | 2023-09-12 | 洛阳普泰生物技术有限公司 | African swine fever virus antibody ELISA detection kit and preparation method thereof |
| CN113607952B (en) * | 2021-08-18 | 2022-03-11 | 杭州恒奥科技有限公司 | African swine fever virus blocking ELISA antibody detection kit and preparation method and application thereof |
| CN115724991A (en) * | 2022-02-23 | 2023-03-03 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Soluble expressed recombinant protein rP22, hybridoma cell strain and application thereof |
| CN115925887B (en) * | 2022-10-27 | 2024-02-09 | 华中农业大学 | African swine fever virus pA104R protein immunodominant B cell epitope, monoclonal antibody thereof and application |
| CN115819564B (en) * | 2022-12-19 | 2023-11-14 | 四川大学 | Single-chain antibody, kit and method for detecting African swine fever |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101818129A (en) * | 2010-04-23 | 2010-09-01 | 天津出入境检验检疫局动植物与食品检测中心 | Hybridoma cell line for anti-African swine fever virus monoclonal antibody and monoclonal antibody secreted thereby |
| CN101831407A (en) * | 2010-04-23 | 2010-09-15 | 天津出入境检验检疫局动植物与食品检测中心 | Hybridoma cell line of monoclonal antibody against African swine fever virus and secreted monoclonal antibody thereof |
-
2014
- 2014-12-05 CN CN201410736335.XA patent/CN104497137B/en not_active Expired - Fee Related
-
2015
- 2015-03-27 WO PCT/CN2015/075183 patent/WO2016086554A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101818129A (en) * | 2010-04-23 | 2010-09-01 | 天津出入境检验检疫局动植物与食品检测中心 | Hybridoma cell line for anti-African swine fever virus monoclonal antibody and monoclonal antibody secreted thereby |
| CN101831407A (en) * | 2010-04-23 | 2010-09-15 | 天津出入境检验检疫局动植物与食品检测中心 | Hybridoma cell line of monoclonal antibody against African swine fever virus and secreted monoclonal antibody thereof |
Non-Patent Citations (2)
| Title |
|---|
| 曹琛福 等: "非洲猪瘟病毒P54蛋白抗原表位预测及鉴定", 《中国预防兽医学报》 * |
| 梁云浩 等: "非洲猪瘟病毒P54蛋白的真核表达及间接ELISA检测方法的建立", 《中国兽医科学》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109810948A (en) * | 2019-01-18 | 2019-05-28 | 中国农业科学院兰州兽医研究所 | The hybridoma cell strain of anti-African swine fever virus K205R protein monoclonal antibody and its antibody of secretion |
| CN109734810A (en) * | 2019-01-24 | 2019-05-10 | 深圳市雅臣智能生物工程有限公司 | Anti- African swine fever virus and CD dual-target pig source antibody, the preparation method and application |
| CN111925436A (en) * | 2019-11-29 | 2020-11-13 | 洛阳普泰生物技术有限公司 | Monoclonal antibody of African swine fever virus P30 protein and application thereof |
| CN111549001A (en) * | 2020-05-28 | 2020-08-18 | 嘉铭(固安)生物科技有限公司 | Hybridoma cell strain secreting African swine fever virus p34 protein monoclonal antibody, monoclonal antibody and application |
| CN111549001B (en) * | 2020-05-28 | 2024-04-19 | 嘉铭(固安)生物科技有限公司 | Hybridoma cell strain secreting African swine fever virus p34 protein monoclonal antibody, monoclonal antibody and application |
| CN111849922B (en) * | 2020-07-20 | 2021-08-03 | 华中农业大学 | Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof |
| CN111849922A (en) * | 2020-07-20 | 2020-10-30 | 华中农业大学 | Monoclonal antibody prepared from African swine fever virus truncated protein p54 and application thereof |
| CN112481220B (en) * | 2020-11-03 | 2021-09-14 | 中国农业科学院兰州兽医研究所 | anti-African swine fever virus helicase D1133L monoclonal antibody, hybridoma cell strain secreting monoclonal antibody and application |
| CN112481220A (en) * | 2020-11-03 | 2021-03-12 | 中国农业科学院兰州兽医研究所 | anti-African swine fever virus helicase D1133L monoclonal antibody, hybridoma cell strain secreting monoclonal antibody and application |
| CN112661817A (en) * | 2020-12-25 | 2021-04-16 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Preparation method, epitope identification and application of African swine fever virus p54 protein monoclonal antibody |
| CN113045673A (en) * | 2021-03-25 | 2021-06-29 | 深圳海关动植物检验检疫技术中心 | African swine fever virus antibody detection kit based on chimeric P54 epitope and application |
| CN113402601A (en) * | 2021-06-09 | 2021-09-17 | 河南中泽生物工程有限公司 | Preparation method and application of anti-African swine fever virus p54 protein monoclonal antibody |
| CN114167055A (en) * | 2021-10-21 | 2022-03-11 | 山东绿都生物科技有限公司 | Competitive enzyme-linked immunosorbent assay kit for detecting anti-African swine fever antibody in serum |
| CN114167055B (en) * | 2021-10-21 | 2023-07-18 | 山东绿都生物科技有限公司 | Competitive enzyme-linked immunosorbent assay kit for detecting anti-African swine fever antibodies in serum |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104497137B (en) | 2017-10-31 |
| WO2016086554A1 (en) | 2016-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104497137A (en) | General monoclonal antibody for African swine fever virus strains as well as preparation method and application thereof | |
| CN104497136A (en) | Monoclonal antibody for African swine fever virus gene II type strain as well as preparation method and application thereof | |
| CN102586193B (en) | Monoclonal antibody for resisting duck tembusu virus, hybridoma strain and application thereof | |
| CN109796531A (en) | Pig Delta coronavirus N protein monoclonal antibody and its epitope and application | |
| CN104086652B (en) | Resisting O-type foot and mouth disease virus specificity single domain antibody and recombinant expression carrier thereof | |
| CN103436496B (en) | The monoclonal antibody of anti-Mycoplasma bovis, the hybridoma cell strain secreting this monoclonal antibody and application | |
| CN104450627B (en) | The monoclonal antibody of 3 type envelope protein ORF132 of Cyprinus carpio herpesviruss, hybridoma cell strain and its application | |
| CN102731615A (en) | Detection reagent and detection method for PRRSV | |
| CN101748136A (en) | Expression of cymbidium mosaic virus coat protein gene and preparation method for antibody of cymbidium mosaic virus coat protein gene | |
| CN106841607B (en) | Acute Hepatopancreatic necrosis syndrome dedicated test kit and preparation method thereof | |
| CN103740649B (en) | The B cell epi-position of anti-blue tongue virus serum 16 type VP2 protein monoclonal antibody BTV16-2B4 and identification thereof and application | |
| CN101921337B (en) | Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application | |
| CN110261606B (en) | Clostridium perfringens beta toxin antibody capture ELISA detection method | |
| CN106866818A (en) | The proteineus yolk antibody of the acute Hepatopancreatic necrosis syndrome of immunodiagnosis and its preparation technology and application | |
| CN106701687A (en) | Hybridoma cell strain and rabies virus phosphoprotein monoclonal antibody generated by same | |
| CN114807178B (en) | African swine fever virus P72 protein C-terminal multi-epitope recombinant antigen and application thereof | |
| CN102621305B (en) | Pig cytomegalovirus antibody indirect blocking enzyme-linked immuno sorbent assay (ELISA) detection kit and detection method thereof | |
| CN108101974A (en) | Fasciola hepatica multi-epitope fusion diagnosis antigen and its application and preparation method | |
| CN104897834B (en) | Method for detecting toxoplasma acute infection and target protein thereof | |
| CN102533666A (en) | Orf virus (ORFV) protein ORFV059 monoclonal antibody hybridoma G3 and monoclonal antibody | |
| CN104965087B (en) | The method and its target proteinses of a kind of efficient detection Infection of Toxoplasma Gondii acute infection | |
| CN103342740B (en) | A kind of blocking ELISA method for detecting fowl HEV specific antibody | |
| CN103305472A (en) | Bluetongue virus serum 1 (BTV1) VP2 protein monoclonal antibody (BTV1-3E8), B-cell epitope polypeptide identified by BTV1-3E8 and application of BTV1-3E8 | |
| CN110794148B (en) | Double-antibody sandwich ELISA quantitative detection kit for pig inflammation small NLRP3 and application thereof | |
| CN116655802B (en) | Fusion protein of melioidosis bacterium Hcp1 protein, monoclonal antibody, hybridoma cell strain and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20171031 Termination date: 20191205 |