CN105368873A - Preparation method, identification method and application of baculovirus for expressing Schmallenberg virus (SBV) recombination N gene - Google Patents
Preparation method, identification method and application of baculovirus for expressing Schmallenberg virus (SBV) recombination N gene Download PDFInfo
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Abstract
The invention discloses a preparation method, an identification method and application of baculovirus for expressing a Schmallenberg virus (SBV) recombination N gene. With reference to Schmallenberg virus disclosed by NCBI: a pair of primers containing specific enzyme digestion sites are designed in the open reading frame sequence of the SBV N gene, and the SBV N gene is amplified; the amplified SBV N gene is cloned in an insect baculovirus expression vector pFastBacHTB, and the like. The preparation method, identification method and application disclosed by the invention have the beneficial effects that SBV nucleoprotein is expressed on an insect cell, finally protein electrophoresis and immunoblotting methods are used for indicating that the experiment obtains SBV N protein having biological activity, and Vbacmid-SBV-N baculovirus is accurately identified.
Description
Technical field
The invention belongs to the preparation of a kind of baculovirus, qualification and applied technical field thereof, be specially a kind of V
bacmidthe preparation of-SBV-N baculovirus, qualification and applied technical field thereof.
Background technology
Execute Maron shellfish lattice virus english abbreviation: SBV, execute Maron shellfish lattice virus disease be a kind of new, be popular in Europe zoonosis.SBV can cause the animal morbidities such as ox, goat, sheep, wild ox, propagates, also by placenta vertical transmission mainly through storehouse midge.Main clinical symptom shows as the clinical symptom such as heating, diarrhoea, weak, stillborn foetus, monster, milk yield decline, newborn cub birth defect.SBV has a strong impact on livestock production performance, harm animal husbandry development.Europe is one of main source area of China's breeding stock introduction and livestock product import.In view of the tremendous economic loss that SBV may bring China's livestock industry, the detection method studying this virus is significant.
Summary of the invention
The present invention passes through by SBVN gene recombination in baculovirus transfer vector gene, and by its successful expression in insect cell.The acquisition of expressing protein is that basic substance has been established in the research of this viral corresponding method of detection.
V in the present invention
bacmid-SBV-N baculovirus refers to the baculovirus of expressing and executing Maron shellfish lattice viral N proteins.
The present invention adopts following technical scheme to realize.
One prepares Vbacmid-SBV-N baculovirus method, and step is as follows:
1) Maron shellfish lattice virus is executed with reference to what NCBI announced: the N gene open reading frame sequence of SBV devises a pair primer containing specific cleavage site, amplification SBVN gene;
2) by amplification SBVN gene clone in baculovirus transfer vector pFastBacHTB;
3) with the Plastid transformation DH10Bac competent cell that step 2 obtains, recombinant shuttle plasmid Bacmid-SBV-N is obtained;
4) recombinant shuttle plasmid Bacmid-SBV-N step 3 obtained, at liposome-mediated lower transfection Sf 9 insect cell, obtains the baculovirus of Vbacmid-SBV-N.
The authentication method of a kind of Vbacmid-SBV-N baculovirus, step is, get Vbacmid-SBV-N baculovirus, Sf9 insect cell is infected in the ratio of 1:1 ~ 1:10, after 27 DEG C of cultivation 96h ~ 120h, get the vial supernatant of cultivation, viruses indentification is carried out: use Bio-Radmini electrophoresis apparatus by SDS-PAGE method, with reference to Bio-Radmini electrophoresis apparatus operational manual specification sheets, SDS-PAGE electrophoresis is carried out to the vial supernatant cultivated, make two clotting glue simultaneously, one piece for staining analysis, another block be used for Westernblot analyze.After SDS-PAGE electrophoresis, protein molecular weight is at 29.6KD place appearance distinctive band.
After carrying out the qualification of expressing viral characteristic protein by SDS-PAGE method, by Westernblot method, expressing viral characteristic protein is identified again: with Bio-Rad transferring film instrument, each protein band of SDS-PAGE gel electrophoresis is transferred on solid phase carrier nitrocellulose filter, with the monoclonal antibody 1F2 (1:1000 doubly dilutes) of anti-SBV nucleoprotein for primary antibodie, sheep anti-mouse igg-HRP (1:3000 doubly dilutes) is that the two anti-Westernblot that are analyze, and protein molecular weight is at 29.6KD place appearance distinctive band.
The primer of a pair preparation Vbacmid-SBV-N baculovirus use, SBVF:5 '-TTAGGATCCATGTCAAGCCAATTCA-3 '; SBVR:5 '-ATTCTGCAGCGTTAGATGTTGATACCGA-3 '.
The restriction enzyme site of the primer of a pair preparation Vbacmid-SBV-N baculovirus use, the restriction enzyme site of SBVF is GGATCC; The restriction enzyme site of SBVR is CTGCAG.
Being applied as of a kind of Vbacmid-SBV-N baculovirus, the nucleoprotein of Vbacmid-SBV-N baculovirus stably express SBV: N protein.
Being applied as of a kind of Vbacmid-SBV-N baculovirus, there is immunological response in the monoclonal antibody of Vbacmid-SBV-N baculovirus and anti-SBVN albumen.
Being applied as of a kind of Vbacmid-SBV-N baculovirus, utilizes the SBV nucleoprotein of Vbacmid-SBV-N baculovirus expression, as the bag in Enzyme-linked Immunosorbent Assay reagent preparation in SBV antibody test by with antigen.
Being applied as of a kind of Vbacmid-SBV-N baculovirus, utilizes the SBV nucleoprotein of Vbacmid-SBV-N baculovirus expression, as in SBV antibody test, and the gold mark material in colloidal gold strip preparation.
Beneficial effect of the present invention is, contriver is better than the advantage of prokaryotic expression system based on insect baculovirus expression system---when expressing foreign protein, there is complete post translational processing Modifying Capability, carried out the expression of SBV nucleoprotein on insect cell.In the process expressing SBV albumen, contriver is in order to control the error that in transfection process, the factor such as reagent, operation causes, when transfection, using the Bacmid-GFP of laboratory structure in early stage as positive control, expression by observing green fluorescent protein after transfection proves the reliability of transfection process, by the method for protein electrophoresis, immunoblotting, final contriver shows that experiment obtains and has bioactive SBVN albumen, identifies V exactly
bacmid-SBV-N baculovirus.
With regard to the drawings and specific embodiments, the present invention is further explained below.
Accompanying drawing explanation
The pcr amplification figure (Fig.1PCRamplificationofrecombinantplasmidSBV-N) of Fig. 1 recombinant plasmid SBV-N
Wherein: M.DNA standard DL2000; 1-4.SBV-N primer SBVF/SBVR amplified fragments (M.DNAMarkerDL2000; 1-4.PCRproductsofrecombinantplasmidSBV-NwithSBVF/SBVRpri mers);
The pcr amplification figure (Fig.2PCRamplificationofrecombinantplasmidpMD18T-SBV-N) of Fig. 2 recombinant plasmid pMD18T-SBV-N
Wherein: M.DNA standard DL2000; 1-2.pMD18T-SBV-N primer SBVF/SBVR amplified fragments (M.DNAMarkerDL2000; 1-2.PCRproductsofrecombinantplasmidpMD18T-SBV-NwithSBVF/ SBVRprimers);
The enzyme of Fig. 3 recombinant plasmid pFastBacHTB-SBV-N cuts qualification figure (Fig.3IdentificationoftherecombinantplasmidofpFastBacHTB-SBV-Nbyenzymedigestion)
Wherein: M.DNA standard MarkerIV; 1.pFastBacHTB-SBV-N is through Bam I and Pst I double digestion (M.DNAMarkerIV; 1.RestrictionenzymeidentificationofrecombinantplasmidpFa stBacHTB-SBV-NbyBam I & Pst I);
The pcr amplification figure (Fig.4PCRamplificationofrecombinantshuttleplasmidBacmid-S BV-N) of Fig. 4 recombinant shuttle plasmid Bacmid-SBV-N
M.DNA standard DL2000; 1-5.Bacmid-SBV-N primer SBVF/SBVR amplified fragments
(M.DNAMarkerDL2000；1-5.PCRproductsofrecombinantplasmidBacmid-SBV-NwithSBVF/SBVRprimers)；
PCR qualification figure (Fig.5IdentificationoftherecombinantplasmidBacmid-SBV-Nby PCR) of Fig. 5 recombinant plasmid Bacmid-SBV-N
Wherein: M.DNA standard DL10000; The 1-11.Bacmid-SBV-N PCR primer of primer M13F and M13R; The 12.Bacmid PCR primer of primer M13F and M13R; (M.DNAMarkerDL10000; 1-11.PCRproductsofrecombinantplasmidBacmid-SBV-NwithM13F andM13Rprimers; 12.PCRproductsofcontrplplasmidBacmidwithM13FandM13Rprime rs);
Fig. 6 recombinant protein expresses figure (10 × 16) (Fig.6ExpressionofrecombinatproteininSf9cell) wherein on Sf9 insect cell: (a). normal Sf9 cell; (b). the Sf9 cell of transfection Bacmid-PPRV-N rod granule; The Sf9 cell of (c) transfection Bacmid-GFP24h; The Sf9 cell of (d) transfection Bacmid-GFP120h; (a) .normalSf9cells; (b) .Sf9cellstransfectedwithBacmid-PPRV-N; (c) .Sf9cellstransfectedwithBacmid-GFPfor24h; (d) .Sf9cellstransfectedwithBacmid-GFPfor120h;
The expression identification figure (Fig.7Identificationofrecombinantprotein) of Fig. 7 recombinant protein
Wherein: M. albumen pre-dyed Marker; 1.Vbacmid-SBV-N (stoste); 2.Vabcmid-SBV-N (stoste); 3. the His-SBV-N albumen (3 times of dilutions) of purifying; 4. the MBP-SBV-N albumen (3 times of dilutions) of purifying
M.Marker；1.Vbacmid-SBV-Nprotein；2.Vabcmid-SBV-Nprotein；3.PurifiedHis-SBV-Nprotein(3timesdilution)；4.PurifiedMBP-SBV-Nprotein(3timesdilution)
(a).SDS-PAGE；(b).Western-blot。
Embodiment
One prepares Vbacmid-SBV-N baculovirus method, and step is as follows:
1) Maron shellfish lattice virus is executed with reference to what NCBI announced: the N gene open reading frame sequence of SBV devises a pair primer containing specific cleavage site, amplification SBVN gene;
2) by amplification SBVN gene clone in baculovirus transfer vector pFastBacHTB;
3) with the Plastid transformation DH10Bac competent cell that step 2 obtains, recombinant shuttle plasmid Bacmid-SBV-N is obtained;
4) recombinant shuttle plasmid Bacmid-SBV-N step 3 obtained, at liposome-mediated lower transfection Sf 9 insect cell, obtains the baculovirus of Vbacmid-SBV-N.
The authentication method of a kind of Vbacmid-SBV-N baculovirus, step is, get Vbacmid-SBV-N baculovirus, Sf9 insect cell is infected in the ratio of 1:1 ~ 1:10, after 27 DEG C of cultivation 96h ~ 120h, get the vial supernatant of cultivation, viruses indentification is carried out: use Bio-Radmini electrophoresis apparatus by SDS-PAGE method, with reference to Bio-Radmini electrophoresis apparatus operational manual specification sheets, SDS-PAGE electrophoresis is carried out to the vial supernatant cultivated, make two clotting glue simultaneously, one piece for staining analysis, another block be used for Westernblot analyze.After SDS-PAGE electrophoresis, protein molecular weight is at 29.6KD place appearance distinctive band.
After carrying out the qualification of expressing viral characteristic protein by SDS-PAGE method, by Westernblot method, expressing viral characteristic protein is identified again: with Bio-Rad transferring film instrument, each protein band of SDS-PAGE gel electrophoresis is transferred on solid phase carrier nitrocellulose filter, with the monoclonal antibody 1F2 (1:1000 doubly dilutes) of anti-SBV nucleoprotein for primary antibodie, sheep anti-mouse igg-HRP (1:3000 doubly dilutes) is that the two anti-Westernblot that are analyze, and protein molecular weight is at 29.6KD place appearance distinctive band.
The primer of a pair preparation Vbacmid-SBV-N baculovirus use, SBVF:5 '-TTAGGATCCATGTCAAGCCAATTCA-3 '; SBVR:5 '-ATTCTGCAGCGTTAGATGTTGATACCGA-3 '.
The restriction enzyme site of the primer of a pair preparation Vbacmid-SBV-N baculovirus use, the restriction enzyme site of SBVF is GGATCC; The restriction enzyme site of SBVR is CTGCAG.
Being applied as of a kind of Vbacmid-SBV-N baculovirus, the nucleoprotein of Vbacmid-SBV-N baculovirus stably express SBV: N protein.
Being applied as of a kind of Vbacmid-SBV-N baculovirus, there is immunological response in the monoclonal antibody of Vbacmid-SBV-N baculovirus and anti-SBVN albumen.
Being applied as of a kind of Vbacmid-SBV-N baculovirus, utilizes the SBV nucleoprotein of Vbacmid-SBV-N baculovirus expression, as the bag in Enzyme-linked Immunosorbent Assay reagent preparation in SBV antibody test by with antigen.
Being applied as of a kind of Vbacmid-SBV-N baculovirus, utilizes the SBV nucleoprotein of Vbacmid-SBV-N baculovirus expression, as in SBV antibody test, and the gold mark material in colloidal gold strip preparation.
Embodiment:
1 materials and methods
1.1 bacterial classifications, plasmid, cell
SBV-N plasmid, monoclonal antibody 1F2 for SBVN albumen, two kinds of SBVN purifying proteins (His-SBV-N: with histidine-tagged fusion rotein His-SBV-N of prokaryotic expression; , in TIANGEN company buy MBP-SBV-N: the fusion rotein MBP-SBV-N of band maltose binding protein tag); PFastBacHTB plasmid, Bacmid-GFP, Top10E.coli and DH10BacE.coli bacterial classification and Sf9 clone are buied in TIANGEN company.
1.2 main agents
Gel recovery test kit and mini-scale plasmid extract test kit purchased from AxyGEN company in a small amount; PrimerSTARHSDNAPloymerase, restriction enzyme BamHI and PstI, T4 ligase enzyme, LATaq enzyme, DNAMarker (DL2000, DL10000) are purchased from precious biotechnology (Dalian) company limited; DNAMarkerIV is purchased from Tian Gen biochemical technology company limited; Protein Marker is purchased from Fermentas company; Foetal calf serum (FBS), Grace ' s insect cell medium, CellfectinII transfection reagent box are purchased from invitrogen company; Ethanol, chloroform, glycerol, Virahol etc. are domestic analytical reagent; EDTA, SDS, beta-mercaptoethanol etc. are purchased from Shanghai bio-engineering corporation; Nutrient broth and nutrient agar medium are purchased from Beijing Luqiao Technology Co., Ltd. of China Import And Export Commodity Inspection Technology Institute; Goat anti-mouse IgG/horseradish enzyme (HRP) mark is purchased from SouthernBiotech company; DAB colouring reagents is purchased from TIANGEN company.
The Design and synthesis of 1.3 primers
Pair of primers is devised for the amplification of N gene and qualification, SBVF:5 '-TTA according to SBV (GenBank accession number :) base sequence
gGATCCaTGTCAAGCCAATTCA-3 '; SBVR:5 '-ATT
cTGCAGcGTTAGATGTTGATACCGA-3 '; Underscore is respectively Bam I and Pst I restriction enzyme site, and the object fragment length of amplification is 722bp.
With reference to " Bac-to-Bac baculovirus expression system service manual ", synthesize the universal primer M13Forward:5 '-GTTTTCCCAGTCACGAC-3 ' for the identification of DH10Bac rod granule; M13Reverse:5 '-CAGGAAACAGCTATGAC-3 '.Primer is synthesized by precious biotech firm.
The amplification of 1.4 goal gene
With SBV-N plasmid for template, SBVF/SBVR is that primer pair SBVN gene carries out specific amplification and obtains object fragment.
The Construction and identification of 1.5 cloning vector pMD18T-SBV-N
The object fragment obtained increasing and pMD18-TVector spend the night in 16 DEG C of water-baths and are connected, coated plate after product conversion to Top10 competent cell will be connected, be inverted flat board incubated overnight in 37 DEG C of constant incubators, single bacterium colony that picking form is good, carry out PCR qualification, positive bacterium liquid is accredited as to PCR and preserves.
The purifying of 1.6 recombinant plasmid pMD18T-SBV-N and expression vector pFastBacHTB
Enlarged culturing after the frozen Top10E.coli containing pMD18T-SBV-N plasmid and the Top10E.coli containing expression vector pFastBacHTB being recovered, adopts the mini-scale plasmid of AxyGEN company to extract test kit and extracts plasmid, in-20 DEG C of preservations.
The Construction and identification of 1.7 restructuring donor plasmid pFastBacHTB-SBV-N
PMD18T-SBV-N plasmid and expression vector pFASTbacHTB plasmid restriction enzyme Bam I and Pst I are carried out double digestion in 37 DEG C, and enzyme is cut rear respectively purifying and is reclaimed, and digestion products connects with T4DNA ligase enzyme 16 DEG C of thermostat water baths and spends the night.Product conversion will be connected to Top10 competent cell, coated plate, be inverted flat board incubated overnight in 37 DEG C of constant incubators, single bacterium colony that picking form is good, carry out PCR qualification, positive bacteria extracting plasmid carries out double digestion qualification, and delivers to the order-checking of precious biotechnology (Dalian) company limited.
The Construction and identification of 1.8 recombinant shuttle plasmid Bacmid-SBV-N
Restructuring donor plasmid pFastBacHTB-SBV-N transforms DH10BacE.coli competent cell, coat on nutrient agar medium (LB) flat board containing kantlex, tsiklomitsin, gentamicin three kinds of microbiotic (Gen, Kan, Tet), be inverted in 37 DEG C of constant incubators and cultivate 48h, single bacterium colony that picking form is good, carries out PCR qualification with SBVF/SBVR primer and M13 primer.
The expression of 1.9 recombinant shuttle plasmids in insect cell
Transfection is carried out with reference to InvitrogenBac-to-BacBaculovirusExpressionSystem operation instruction handbook, by the Sf9 insect cell of recombinant shuttle plasmid Bacmid-SBV-N and Bacmid-GFP in liposome-mediated lower transfection logarithmic phase growth, wherein Bacmid-GFP transfection is as positive control.Cultivate in 28 DEG C of cell culture incubators, the growing state of a cell is observed every 24h, according to cell growth status in about 96h ~ 120h collecting cell nutrient solution, multigelation cell 3 times, the centrifugal 10min of 3000rpm/min, supernatant liquor is recombinant baculovirus liquid.Obtaining s-generation virus through going down to posterity, analyzing for SDS-PAGE and Westernblot, can by its packing when result is positive, frozen work kind poison.
The SDS-PAGE qualification of 1.10 recombinant proteins and Westernblot analyze
1.10.1SDS-PAGE qualification
Use Bio-Radmini electrophoresis apparatus, with reference to Bio-Radmini electrophoresis apparatus operational manual specification sheets, SDS-PAGE electrophoresis carried out to recombinant protein sample, makees two clotting glue simultaneously, one piece for staining analysis, another block is used for Westernblot and analyzes.The gel separated first uses distilled water flushing, then soaks gel with Coomassie brilliant blue staining fluid, is placed on room temperature dyeing 1h on shaking table.After having dyeed, with the decolouring of Coomassie brilliant blue destainer to background water white transparency, observe electrophoresis result under natural light, judge whether target protein expresses, photographic recording experimental result with reference to protein Maker and negative control.The qualification result of this step is, whether this baculovirus can express SBVN albumen.
1.10.2Westernblot Analysis and Identification
With Bio-Rad transferring film instrument, each protein band of SDS-PAGE gel electrophoresis is transferred on solid phase carrier nitrocellulose filter, with the monoclonal antibody 1F2 (1:1000 doubly dilutes) of anti-SBV nucleoprotein for primary antibodie, sheep anti-mouse igg-HRP (1:3000 doubly dilutes) two anti-does Westernblot Analysis and Identification.The qualification result of this step is that this albumen has biologic activity or no.
2 experimental results
The amplification of 2.1 goal gene
With primer SBVF/SBVR, SBV-N plasmid is increased, occur single band (Fig. 1) at 722bp place, prove to obtain the goal gene fragment needed for clone.
2.2 the qualification of cloning vector pMD18T-SBV-N
Recombinant plasmid pMD18T-SBN-N primer SBVF/SBVR carries out PCR preliminary evaluation, and amplified production is 722bp (Fig. 2), conforms to theoretical value.
The qualification of 2.3 restructuring donor plasmid pFastBacHTB-SBV-N
Recombinant plasmid pFastBacHTB-SBV-N can cut out the SBVN goal gene band (Fig. 3) of 722bp through Bam I and Pst I double digestion, size conforms to expected results, the external source object clip size that further sequencing result confirmation is inserted and reading frame correctly, prove successfully to construct restructuring donor plasmid pFastBacHTB-SBV-N.
The PCR qualification of 2.4 recombinant shuttle plasmid Bacmid-SBV-N
After pFastBacHTB-SBV-N proceeds to DH10Bac competent cell, after the screening of Gen, Kan, Tet3 kind microbiotic plate, extract its DNA, PCR qualification is carried out with primer SBVF/SBVR, amplified production is 722bp (Fig. 4), carry out PCR qualification with M13 forward and reverse primer simultaneously, amplified production is 3152bp, the DH10Bac bacterial strain of unconverted plasmid amplifies the object band (Fig. 5) of 300bp, this result proves object fragment, and swivel base is to Bacmid, and recombinant shuttle plasmid Bacmid-SBV-N successfully constructs.
The expression of 2.5 recombinant proteins in insect cell
Extract swivel base bacmid dna, at the transfection Sf 9 insect cell of liposome-mediated lower transfection logarithmic phase growth, positive control time simultaneously using the Bacmid-GFP of expressing green fluorescent protein as transfection, cultivate about 48h visible cell pathology under inverted microscope for 28 DEG C, compared with normal cell, transfectional cell becomes large, becomes circle (Fig. 6 (a) and (b)).The fluorescin (Fig. 6 (c)) that the insect cell 24h of transfection Bacmid-GFP sees, 120h after transfection, the cell of express fluorescent protein reaches more than 80% (Fig. 6 (d)), according to the expression of fluorescin, the best virus harvest time can be estimated.
The expression identification of 2.6 recombinant proteins
By the sf9 insect cell that recombinant shuttle plasmid Bacmid-SBV-N and positive control Bacmid-GFP transfection logarithmic phase grow, in 28 DEG C of cell culture incubators, cultivate about 96h ~ 120h collecting cell nutrient solution as kind of a poison.The s-generation virus (Vbacmid-SBV-N) obtained through going down to posterity identifies the expression of SBV nucleoprotein.SDS-PAGE and Westernblot qualification result all shows, recombinant protein successful expression (Fig. 7 (a, b)).
Claims (9)
1. prepare V for one kind
bacmid-SBV-N baculovirus method, it is characterized in that, step is as follows:
1) Maron shellfish lattice virus is executed with reference to what NCBI announced: the N gene open reading frame sequence of SBV devises a pair primer containing specific cleavage site, amplification SBVN gene;
2) by amplification SBVN gene clone in baculovirus transfer vector pFastBacHTB;
3) with the Plastid transformation DH10Bac competent cell that step 2 obtains, recombinant shuttle plasmid Bacmid-SBV-N is obtained;
4) recombinant shuttle plasmid Bacmid-SBV-N step 3 obtained, at liposome-mediated lower transfection Sf 9 insect cell, obtains V
bacmidthe baculovirus of-SBV-N.
2. a V
bacmidthe authentication method of-SBV-N baculovirus, is characterized in that, gets V
bacmid-SBV-N baculovirus, Sf9 insect cell is infected in the ratio of 1:1 ~ 1:10, after 27 DEG C of cultivation 96h ~ 120h, get the vial supernatant of cultivation, carry out viruses indentification by SDS-PAGE method: use Bio-Radmini electrophoresis apparatus, with reference to Bio-Radmini electrophoresis apparatus operational manual specification sheets, SDS-PAGE electrophoresis is carried out to the vial supernatant cultivated, make two clotting glue simultaneously, one piece for staining analysis, another block be used for Westernblot analyze; After SDS-PAGE electrophoresis, protein molecular weight is at 29.6KD place appearance distinctive band.
3. a kind of V according to claim 2
bacmidthe authentication method of-SBV-N baculovirus, it is characterized in that, after carrying out the qualification of expressing viral characteristic protein by SDS-PAGE method, by Westernblot method, expressing viral characteristic protein is identified again: with Bio-Rad transferring film instrument, each protein band of SDS-PAGE gel electrophoresis is transferred on solid phase carrier nitrocellulose filter, with the monoclonal antibody 1F2 (1:1000 doubly dilutes) of anti-SBV nucleoprotein for primary antibodie, sheep anti-mouse igg-HRP (1:3000 doubly dilutes) is that the two anti-Westernblot that are analyze, protein molecular weight is at 29.6KD place appearance distinctive band.
4. a pair preparation V
bacmidthe primer that-SBV-N baculovirus uses, is characterized in that, SBVF:5 '-TTAGGATCCATGTCAAGCCAATTCA-3 '; SBVR:5 '-ATTCTGCAGCGTTAGATGTTGATACCGA-3 '.
5. a pair preparation V according to claim 4
bacmidthe restriction enzyme site of the primer that-SBV-N baculovirus uses, it is characterized in that, the restriction enzyme site of SBVF is GGATCC; The restriction enzyme site of SBVR is CTGCAG.
6. a V
bacmidthe application of-SBV-N baculovirus, is characterized in that, this is applied as, V
bacmidthe nucleoprotein of-SBV-N baculovirus stably express SBV: N protein.
7. a V
bacmidthe application of-SBV-N baculovirus, is characterized in that, this is applied as, V
bacmidthere is immunological response in the monoclonal antibody of-SBV-N baculovirus and anti-SBVN albumen.
8. a V
bacmidthe application of-SBV-N baculovirus, is characterized in that, this is applied as, utilizes V
bacmidthe SBV nucleoprotein of-SBV-N baculovirus expression, as the bag in Enzyme-linked Immunosorbent Assay reagent preparation in SBV antibody test by with antigen.
9. a V
bacmidthe application of-SBV-N baculovirus, is characterized in that, this is applied as, utilizes V
bacmidthe SBV nucleoprotein of-SBV-N baculovirus expression, as in SBV antibody test, the gold mark material in colloidal gold strip preparation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107991494A (en) * | 2017-11-22 | 2018-05-04 | 贾赟 | A kind of liquid phase protein chip kit of the external and new hair animal epidemic of while four kinds of detection and preparation method thereof |
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