CN101724652A - Building method of silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system - Google Patents
Building method of silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system Download PDFInfo
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Abstract
The invention discloses a building method of a silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system, comprising: taking silkworm wild type BmNPV genome DNA as the template; respectively taking P10-upF/P10-upB and P10-downF/P10-down B as a primer PCR for amplification to obtain p10-up and p10-down; after processed, inserting into BamHI-PstI-HindIII locus in pUC 19 to obtain recombinant plasmid pUC19-p10-up-down; using pUC-19-p10-up-polh-down, liposome lipofectin and MilliQ H2O to prepare DNA-Lipofectin mixed liquor; adding the mixed liquor into BmN cells for cultivating; collecting transfection cell supernatant; inoculating BmN cells, and recovering a polyhedral body; extracting virus DNA from the polyhedral body and electrically converting DH10 beta competent cells; screening locus ceruleus and cultivating; after cultivating PCR positive bacterial plaque, extracting macro-molecular DNA to transfect to the BmN cells; separating to obtain helper plasmids from DH10Bac culture bacteria of AcMNPV Bac-to-Bac; converting the helper plasmids into E.coli DH10 beta containg Ploh+BmBacmid; and screening the DH10 beta bacterial strain of the helper plasmid containg Ploh+BmBacmid. The invention can produce recombinant virus capable of infecting by eating with mouth, and recombinant virus does not need to infect by intracutaneous inoculation, thus improving the production efficiency of the silkworm rhabdovirus expression system.
Description
Technical field
The present invention relates to the baculovirus gene expression system in the biotechnology, relate in particular to a kind of silkworm BmNPV Polh
+The construction process of Bac-to-Bac baculovirus expression system.
Background technology
Silkworm is the economic resources insect of China's number of animals raised maximum, and along with the development of modern biotechnology, the exploitation silkworm has the prospect of marketing preferably as new-type bioreactor.
The silkworm baculovirus gene expression system is one of eukaryotic expression system efficiently at present.The Bac-to-Bac baculovirus expression system that we utilize the bacterial transposon principle to make up to be specifically designed to silkworm (referring to the granted patent ZL200310108781.8 before us).The advantage of this system is that recombinant baculovirus can be by the assignment of genes gene mapping transferance of bacterial transposon, in intestinal bacteria, realize the transfer reorganization of gene, obtain recombinant virus fast, made up the Bac-to-Bac rapid gene expression system that can utilize China characteristic resources insect-silkworm.This system by donor plasmid, contain the genomic competence bacterium of silkworm baculovirus BmBacmid DH10BmBac and constitute, its principle that produces recombinant virus is: at first the external source goal gene is cloned into donor plasmid, donor plasmid is transformed in the DH10BmBac competent cell then, fixed point transposition effect by bacterial transposon, promotor and goal gene are changed over to the silkworm baculovirus genome in the lump and produce recombinant virus dna, last transfection silkworm cultured cell and obtain to contain the goal gene recombinant virus.But the recombinant virus that said system makes up all is strong promoter-polyhedron promotor of utilizing baculovirus and the recombinant baculovirus that makes up, and all generally are polyhedron absence type (polh
-) virus, such recombinant virus can't peroral infection, therefore can only realize the expression of infection and external source goal gene when infected silkworm by method loaded down with trivial details, percutaneous injection, influence the expression efficiency of silkworm baculovirus greatly.This has become a bottleneck problem being badly in need of solution in the silkworm biological reactor mass-producing application.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can express polyhedrosis silkworm BmNPV Polh simultaneously
+The construction process of Bac-to-Bac baculovirus expression system.Utilize the recombinant virus of this system's generation to add food realization infection expression by per os, better having solved recombinant virus must improve the production efficiency of baculovirus expression vector system through the defective of bark graft kind infection.
In order to achieve the above object, the contriver is that ZL200310108781.8, name are called on the basis of Chinese patent of " utilizing bacterial transposon to make up the method for silkworm virus rapid gene expression system " in the patent No., utilize homologous recombination technique that polyhedron polh gene is imported p10 site in the BmNPV Bacmid genome, made up and to have produced polyhedrosis Bac-to-Bac system (with Polh
+Bac-to-Bac represents).
Technical scheme of the present invention is as follows:
This silkworm BmNPV Polh
+The step of the construction process of Bac-to-Bac baculovirus expression system is:
1) structure of recombinant transfer vector:
With silkworm wild-type BmNPV genomic dna is template, be flanking sequence p10-up and the p10-down that the primer PCR amplification obtains p10 gene each 2kb of upstream and downstream with P10-upF/P10-upB and P10-downF/P10-downB respectively, described p10-up comprises the p10 promotor; The designed restriction enzyme site of primer is used the underscore mark in table; Described pcr amplification condition is: 94 ℃ of sex change 3min, carry out 30 circulations in proper order by 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ of extension 2min, and last 72 ℃ are extended 7min; P10-up and p10-down fragment that amplification obtains are used ethanol sedimentation, purification process earlier, cut processing with HindIII/PstI and PstI/BamHI enzyme respectively again, then enzyme is cut the p10-up that handled and p10-down fragment and successively be inserted into BamHI-PstI-HindIII site among the pUC19, obtain containing p10 gene flanking sequence recombinant plasmid pUC19-p10-up-down from beginning to end;
With silkworm wild-type BmNPV genomic dna is template, is that the primer PCR amplification obtains the polyhedrosis gene fragment with PolhF/PolhB; The pcr amplification reaction condition is: 94 ℃ of sex change 3min, carry out 30 circulations in proper order by 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ of extension 1min, and last 72 ℃ are extended 7min; The polyhedrosis gene fragment that the clone obtains is cut the Pst I site that connects into pUC19-p10-up-down after the processing with Pst I enzyme, with the plasmid called after pUC19-p10-up-polh-down of forward insertion wherein;
2) generation of Polh+BmBacmid and screening:
In transparent polypropylene sterile tube, add 10 μ L concentration and be the pUC19-p10-up-polh-down of 1 μ g/ μ L, BmNPV bacmid DNA, 14 μ L liposome lipofectin that 1 μ L concentration is 1 μ g/ μ L and the MilliQ H of 15 μ L sterilization
2O, mixing is placed 15min under the normal temperature gently, obtains the DNA-Lipofectin mixed solution; The BmN cell that is in logarithmic phase in the culture dish is washed 3 times with serum-free TC-100 substratum, and the back adds 2mL serum-free TC-100 substratum; Then described DNA-Lipofectin mixed solution is evenly joined in the BmN cell, 27 ℃ of overnight incubation are inhaled and to be removed supernatant, add 2mL fresh contain serum TC-100 substratum, cultivated 5 days for 27 ℃;
Cell conditioned medium after the collection transfection is inoculated the BmN cell subsequently, reclaims polyhedron; Extracting viral DNA from the polyhedron of collecting; Then this DNA electricity is transformed DH10 β competent cell, on the culture plate that contains kantlex, X-gal and IPTG, screen locus coeruleus; After cultivating 48h, choose spot and carry out the PCR evaluation, analyze polyhedrosis gene and whether exist; To extracting macromole DNA wherein after the PCR male bacterial plaque incubated overnight, whether further electrophoresis is identified to observe has transferring plasmid to exist among this macromole DNA, then the macromole DNA that obtains is transfected into the BmN cell, observes this BmN cell and whether infect and produce polyhedron; Whether the recombinant virus that the polyhedron of purifying is added food silkworm observation generation has peroral infection;
3) silkworm BmNPV Polh
+The structure of Bac-to-Bac expression system:
Separating the acquisition size with common plasmid extraction method from the DH10Bac cultivation bacterium of AcMNPV Bac-to-Bac is the helper plasmid of 13.2kb; This helper plasmid is transformed into contains Polh
+Among the E.coliDH10 β of BmBacmid, screening contains Polh simultaneously on the culture plate that contains kantlex and tsiklomitsin
+The DH10 β bacterial strain of BmBacmid and helper plasmid.
Compared with prior art, the invention has the beneficial effects as follows: utilize bacterial transposon locus specificity swivel base principle to make up and to form polyhedrosis recombinant silkworm virus, such recombinant virus can exist with polyhedron and two kinds of forms of blastogenesis C-type virus C, can carry out small-scale easily in culturing cell expresses, can utilize the direct peroral infection larva of polyhedron to realize large-scale production again, for the silkworm biological reactor industrialization provides strong technical support.
Description of drawings
Fig. 1 forward inserts the structure iron of plasmid pUC19-p10-up-polh-down;
Fig. 2 DH10BmBac (polh
+) the structure schema;
Fig. 3 silkworm Polh
+BmBacmid DNA transfection BmN cell and SDS-PAGE analyze;
Wherein, (A) Polh
+BmBacmid DNA transfection BmN cell 72h can be observed a large amount of polyhedroies in the cell; (B) protein analysis of cells infected, 1 is contrast, i.e. BmBacmid (polh
-) virus infection, 2 is Polh
+The BmBacmid virus infection, arrow is depicted as polyhedrin, and molecular weight is about 29kD, and the standard molecular weight mark is as shown in the figure;
Fig. 4 silkworm BmNPV Polh
+Bac-to-Bac expression alien gene EGFP;
Wherein, (A), (B) be vBmBac (the polh+)-photo of EGFP infected B mN cell under same visual field, A) is that visible light is taken, (B) be the fluorescence photo after blue-light excited; (C) be vBmBac (polh
+The SDS-PAGE collection of illustrative plates of)-EGFP infected B mN cell, arrow are depicted as polyhedrin and EGFP albumen (molecular weight is very approaching, is difficult to distinguish).D. be vBmBac (polh
+The Western blot of)-EGFP infected B mN cell analyzes.
Embodiment
1) structure of recombinant transfer vector:
With silkworm wild-type BmNPV genomic dna is template, is flanking sequence p10-up (comprising the p10 promotor) and the p10-down that primer (table 1) PCR obtains each about 2kb of p10 gene upstream and downstream with P10-upF/P10-upB and P10-downF/P10-downB respectively.The designed restriction enzyme site of primer is used the underscore mark in table.The pcr amplification condition is: 94 ℃ of sex change 3min; Below carry out 30 circulations: 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 2min; Last 72 ℃ are extended 7min.After ethanol sedimentation, the purification process, p10-up and p10-down fragment that amplification obtains are cut the BamHI-PstI-HindIII site of handling and successively inserting among the pUC19 with HindIII/PstI and PstI/BamHI enzyme respectively, obtain containing p10 gene flanking sequence recombinant plasmid pUC19-p10-up-down from beginning to end.
With silkworm wild-type BmNPV genomic dna is template, is that primer PCR obtains the polyhedrosis gene fragment with PolhF/PolhB.The pcr amplification reaction condition is: 94 ℃ of sex change 3min, carry out 30 circulations by 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ of orders of extending 1min, and last 72 ℃ are extended 7min.The fragment that the clone obtains is cut the PstI site that connects into pUC19-p10-up-down after the processing with the PstI enzyme, and with the plasmid called after pUC19-p10-up-polh-down of forward insertion wherein, its structure as shown in Figure 1.
The primer that table 1 the present invention relates to
| Primer | Sequence (5 '-3 ') | Size/bp |
| ??P10-upF??P10-upB??P10-downF??P10-downB??PolhF??PolhB | ??AAAGTCTATTG AAGCTTACGAG(Hind?III)??GTAA CTGCAGTGTAATTTACAG(Pst?I)??ATCAATTGTT CTGCAGTATTCG(Pst?I)??ACA GGATCCGATTTAACTAATG(BamH?I)??TAA CTGCAGCTATAAATATGCC(Pst?I)??ATGTA CTGCAGACAATGTATAG(Pst?I) | ??1894??1998??780 |
2) Polh
+The generation of BmBacmid and screening:
The MilliQ H that in transparent polypropylene sterile tube, adds 10 μ L pUC19-p10-up-polh-down (1 μ g/ μ L), 1 μ L BmNPV bacmid DNA (1 μ g/ μ L), 14 μ L liposome lipofectin and 15 μ L sterilization
2O (amounting to 40 μ L), mixing is placed 15min under the normal temperature gently.During this time the BmN cell that is in logarithmic phase in the d=35mm culture dish is washed 3 times with serum-free TC-100 substratum, the back adds 2mL serum-free TC-100 substratum.Then above-mentioned DNA-Lipofectin mixed solution is evenly added in the cell, 27 ℃ of overnight incubation are inhaled and to be removed supernatant, add 2mL fresh contain serum TC-100 substratum, cultivate 5d for 27 ℃.
Cell conditioned medium after the collection transfection is inoculated the BmN cell subsequently, reclaims polyhedron.Extracting viral DNA from the polyhedron of collecting.Then the DNA electricity is transformed DH10 β competent cell, on the culture plate that contains kantlex, X-gal and IPTG, screen locus coeruleus.After cultivating 48h, choose spot and carry out the PCR evaluation, analyze polyhedrosis gene and whether exist.Extracting macromole DNA wherein after the PCR male bacterial plaque incubated overnight, whether further electrophoresis is identified and is observed the existence whether transferring plasmid is wherein arranged, and then the macromole DNA that obtains is transfected into the BmN cell, observe to infect and produce polyhedron.Whether the recombinant virus that the polyhedron of purifying is added food silkworm observation generation has peroral infection.Polh
+BmBacmid makes up flow process as shown in Figure 2.
3) silkworm BmNPV Polh
+The structure of Bac-to-Bac expression system:
Separating the acquisition size with common plasmid extraction method from the DH10Bac cultivation bacterium (interpolation tsiklomitsin) of AcMNPV Bac-to-Bac (Invitrogen) is the helper plasmid of 13.2kb.It is transformed into contains Polh
+Among the E.coli DH10 β of BmBacmid, screening contains Polh simultaneously on the culture plate that contains kantlex and tsiklomitsin
+The DH10 β bacterial strain of BmBacmid and helper plasmid.
4) silkworm BmNPV Polh
+The applicable cases checking of Bac-to-Bac baculovirus expression system:
Made up the recombinant virus of expressing green fluorescent protein EGFP.At first reporter gene EGFP is inserted the EcoR I site among the donor plasmid pFastBacHT,, transform the Polh that contains above-mentioned structure simultaneously according to the method for Bac-to-Bac construction of recombinant virus
+The DH10 β competent cell of BmBacmid and helper plasmid, 4h (this process producer swivel base) is cultivated in concussion on the LB liquid nutrient medium, get part dilution bacterium liquid coated plate then, utilize kantlex, gentamicin, tsiklomitsin, X-gal and IPTG screening to obtain Polh
+The bacterial strain (white bacterial plaque) that contains EGFP in the BmBacmid genome.
The generation of recombinant virus and expression: choose hickie cultivation and extraction Bacmid genome wherein, transfection BmN cell.Inoculation about 9 * 10 on the d=35mm culture dish
5The BmN culturing cell, place 1h for 27 ℃ and make cell attachment.Solution below preparing in the aseptic pipe of 12mm * 75mm: solution A, the baculovirus DNA that 5 μ L prepare in a small amount are dissolved into the serum-free TC-100 substratum of 100 μ L; Solution B, 6 μ LCellfectin agent dissolves are to 100 μ L serum-free TC-100 substratum.With A, B solution mixing, room temperature is placed 30min.During this time adherent BmN cell is washed 1 time with the 2mL serum free medium, added the 1mL serum free medium again.Add above-mentioned lipid-DNA mixture then.Remove transfection mixture behind the 5h, add the TC-100 that 2mL contains 10%FBS.Results are viral after cultivating 96h in 27 ℃ of incubators.So just obtained to express simultaneously the recombinant virus of polyhedrin and EGFP.Collect the transfection supernatant, the blastogenesis C-type virus C that obtains can be used for infecting the BmN cell.
The SDS-PAGE of expressing protein and Western blot analyze: expressing protein carries out SDS-PAGE and analyzes (showing as Fig. 3), the result shows, the a large amount of polyhedroies that produce, and foreign gene has obtained high-caliber expression, subsequently albumen is transferred to pvdf membrane, that uses one anti-is mouse-anti His monoclonal antibody, two anti-are the anti-mouse IgG of HRP labelled goat antibody, use the TMB colour developing at last, carry out Western blot and detect (showing) as Fig. 4, the result shows that the new system that makes up in this patent not only can form polyhedron, and foreign gene also can access the expression of higher level.
Virus peroral infection silkworm larva condition survey: infect the BmN cell with metainfective supernatant liquor, collection, purifying polyhedron.The polyhedron of purifying is suspended in the PBS damping fluid, measures polyhedron concentration, and to be diluted to concentration with the PBS damping fluid be 3.0 * 10 with blood counting chamber
10NPB/mL.Freshening the food method is: mulberry leaf are cut into the square small pieces of 2cm, evenly smear the viral liquid of 30 μ L on every leaf, feed after dried slightly and play silkworm 4 ages, every silkworm is fed 1 and is with malicious blade, treat that silkworm will be with malicious leaf to get food to finish after, raise according to a conventional method.Smear blade as blank with the aseptic PBS of 30 μ L.Simultaneously with wild-type BmNPV as positive control.Observe the silkworm infection conditions behind the 45d, the result shows, finds that 100% individuality all shows tangible infection symptoms, and is identical with the situation that wild-type virus infects.The result shows that the ODV virus particle has been forgiven in the polyhedron inside of formation, such recombinant virus can be realized infecting by per os.
Claims (1)
1. silkworm BmNPV Polh
+The construction process of Bac-to-Bac baculovirus expression system is characterized in that the step of this method is as follows:
1) structure of recombinant transfer vector:
With silkworm wild-type BmNPV genomic dna is template, be flanking sequence p10-up and the p10-down that the primer PCR amplification obtains p10 gene each 2kb of upstream and downstream with P10-upF/P10-upB and P10-downF/P10-downB respectively, described p10-up comprises the p10 promotor; The designed restriction enzyme site of primer is used the underscore mark in table; Described pcr amplification condition is: 94 ℃ of sex change 3min, carry out 30 circulations by 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ of extension 2min, and last 72 ℃ are extended 7min; P10-up and p10-down fragment that amplification obtains are used ethanol sedimentation, purification process earlier, cut processing with HindIII/PstI and PstI/BamHI enzyme respectively again, then enzyme is cut the p10-up that handled and p10-down fragment and successively be inserted into BamHI-PstI-HindIII site among the pUC19, obtain containing p10 gene flanking sequence recombinant plasmid pUC 19-p10-up-down from beginning to end;
With silkworm wild-type BmNPV genomic dna is template, is that the primer PCR amplification obtains the polyhedrosis gene fragment with PolhF/PolhB; The pcr amplification reaction condition is: 94 ℃ of sex change 3min, carry out 30 circulations by 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ of extension 1min, and last 72 ℃ are extended 7min; The polyhedrosis gene fragment that the clone obtains is cut the PstI site that connects into pUC19-p10-up-down after the processing with the PstI enzyme, with the plasmid called after pUC19-p10-up-polh-down of forward insertion wherein;
2) generation of Polh+BmBacmid and screening:
In transparent polypropylene sterile tube, add pUC19-p10-up-polh-down that μ L concentration is 1 μ g/ μ L, BmNPV bacmid DNA, 14 μ L liposome lipofectin that 1 μ L concentration is 1 μ g/ μ L and the MilliQ H of 15 μ L sterilization
2O, mixing is placed 15min under the normal temperature gently, obtains the DNA-Lipofectin mixed solution; The BmN cell that is in logarithmic phase in the culture dish is washed 3 times with serum-free TC-100 substratum, and the back adds 2mL serum-free TC-100 substratum; Then described DNA-Lipofectin mixed solution is evenly joined in the BmN cell, 27 ℃ of overnight incubation are inhaled and to be removed supernatant, add 2mL fresh contain serum TC-100 substratum, cultivated 5 days for 27 ℃;
Cell conditioned medium after the collection transfection is inoculated the BmN cell subsequently, reclaims polyhedron; Extracting viral DNA from the polyhedron of collecting; Then this DNA electricity is transformed DH10 β competent cell, on the culture plate that contains kantlex, X-gal and IPTG, screen locus coeruleus; After cultivating 48h, choose spot and carry out the PCR evaluation, analyze polyhedrosis gene and whether exist; To extracting macromole DNA wherein after the PCR male bacterial plaque incubated overnight, whether further electrophoresis is identified to observe has transferring plasmid to exist among this macromole DNA, then the macromole DNA that obtains is transfected into the BmN cell, observes this BmN cell and whether infect and produce polyhedron; Whether the recombinant virus that the polyhedron of purifying is added food silkworm observation generation has peroral infection;
3) silkworm BmNPV Polh
+The structure of Bac-to-Bac expression system:
Separating the acquisition size with common plasmid extraction method from the DH10Bac cultivation bacterium of AcMNPV Bac-to-Bac is the helper plasmid of 13.2kb; This helper plasmid is transformed into contains Polh
+Among the E.coliDH10 β of BmBacmid, screening contains Polh simultaneously on the culture plate that contains kantlex and tsiklomitsin
+The DH10 β bacterial strain of BmBacmid and helper plasmid.
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