CN101724651A - Dual-host recombination rhabdovirus expression vector and construction method and application thereof - Google Patents
Dual-host recombination rhabdovirus expression vector and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a dual-host recombination rhabdovirus expression vector and a construction method and application thereof, belonging to the technical field of gene engineering. The dual-host recombination rhabdovirus expression vector is provided with a recognizable Polyhedrin promoter of an insect cell and a recognizable CMV-IE promoter of an animal cell. Dual-host recombination rhabdovirus can be used for preparing a functional protein on the insect cell or can express a target foreign protein on the animal cell and can be used for gene therapy and nonreplication vector vaccine. The construction method of the dual-host recombination rhabdovirus expression vector comprises the following steps of: taking a transfer vector pFas in a Bac-to-Bac expression system as a base; introducing a CMV-IE promoter expression kit on the upstream of the Polyhedrin promoter; constructing the transfer vector; transforming a DH10Bac competent cell; recombining a dual-host Bacmid shuttle vector; and then transfecting insect cells in a logarithmic phase to obtain the dual-host recombination rhabdovirus expression vector.
Description
Technical field
The present invention relates to genetic engineering technique, insect baculovirus gene expression system, gene therapy and live vector vaccine carrier, be specifically related to a kind of dual-host recombination rhabdovirus expression vector and construction process thereof and application.
Background technology
The insect baculovirus gene expression system has superpower expression efficiency; Expression product is had the translation post-treatment modify ability; Can keep foreign protein to bring into play the necessary space structure of its biologic activity; Can not in zooblast, duplicate the biological safety height; Pair cell does not have toxicity basically; Simple to operate, preparation easily; Inserting characteristics and advantages such as the foreign gene capacity is big, is one of current the most widely used eukaryotic expression system.
American I nvitrogen company has released the Bac-to-Bac expression system.The shuttle vectors Bacmid of Bac-to-Bac system contains F-factor replicon (can duplicate), kalamycin resistance gene and Tn7 swivel base site attTn7 in intestinal bacteria.The transfer vector of Bac-to-Bac system is a series of commercial plasmids, comprises the pFastBac I that expresses non-fusion rotein, the pFastBacHTa of expressed fusion protein, b, c and binary vector pFastBacDual.The common feature of these several plasmids is all to contain mimi-Tn7 element sequences, polyhedrin gene promoter (also containing the p10 gene promoter among the pFastBacDual), multiple clone site and as the gentamicin resistant gene of transformation marker.Transfer vector pFastBacDual is a binary expression vector, basic structure and pFastBacI are similar, difference be between the left and right arms of Tn7 except have the Polyhedrin promotor with and subsequent the external upstream of polyclone restriction enzyme site (MSC) sequence also have a reverse p10 gene promoter and polyclone restriction enzyme site (MSC) sequence.Use this carrier simultaneously two kinds of foreign genes to be introduced the recombinant baculovirus genome and to realize coexpression (title: a kind of animal genetic engineering interferon alpha and gamma composite preparations and production method thereof and clinical application, application number: 200610128462.7).The Bac-to-Bac system compares with traditional baculovirus vector system, two big advantages are arranged: the one, shorten recombinant virus greatly and made up the required time, even traditional method person skilled in the art also needed for 6～9 week even longer times, and use the Bac-to-Bac strategy with just obtaining required reorganization bar virus-virus in 7～9 days; The 2nd, because recombinant virus dna produces in bacterium, can be blue hickie screening according to the bacterial plaque dithering, not have the problem of wild-type and non-recombinant type virus crossed contamination, therefore not need the loaded down with trivial details plaque analysis of tradition to come purification of Recombinant virus, and swivel base rate height, the purifying rate reaches 100%.Therefore the Bac-to-Bac expression system is to use expression system comparatively widely at present.
Insect baculovirus is mainly bred on insect cell, and can not in zooblast, duplicate, pair cell does not have toxicity basically, therefore can be used for gene therapy carries and nonreplication vector vaccine (title: a kind of preparation method who can be used for making the recombinant baculovirus of bird vaccine, application number: 200610009656.5).
Be used for when the recombinant baculovirus of expression alien gene on the insect cell is being expressed foreign protein, recombinant baculovirus is also bred in a large number, produce the baculovirus of high-gradient, these recombinant baculovirus are except that staying a part once more as planting the poison the no any utility values of great majority.And as the recombinant baculovirus of gene therapy and nonreplication vector vaccine, need on insect cell, carry out high-gradient propagation, extract recombinant baculovirus then and be used for gene therapy and nonreplication vector vaccine, but do not express foreign protein in the process that recombinant baculovirus is bred on insect cell.Therefore both develop can be on insect cell expression alien gene, simultaneously again can be significant as the recombination rhabdovirus expression vector of gene therapy and nonreplication vector vaccine.
Summary of the invention
The object of the present invention is to provide a kind of dual-host recombination rhabdovirus expression vector, this recombination rhabdovirus expression vector contains discernible Polyhedrin promotor of insect cell and the discernible CMV-IE promotor of zooblast simultaneously, this dual-host recombination rhabdovirus expression vector can the high level expression foreign gene on insect cell, recombinant baculovirus is bred in a large number simultaneously, the recombinant baculovirus of high-gradient breeding can be used as gene therapy and nonreplication vector vaccine, expression alien gene on animal and cell thereof again.
Another object of the present invention is to provide a kind of construction process and application thereof of dual-host recombination rhabdovirus expression vector.
The objective of the invention is to be achieved through the following technical solutions:
Dual-host recombination rhabdovirus expression vector of the present invention, this recombination rhabdovirus expression vector contain discernible Polyhedrin promotor of insect cell and the discernible CMV-IE promotor of zooblast simultaneously.
Dual-host recombination rhabdovirus expression vector of the present invention also contains the discernible p10 promotor of insect cell.
The construction process method of dual-host recombination rhabdovirus expression vector of the present invention may further comprise the steps:
(1) structure of recombinant baculovirus transfer vector:
(A) based on transfer vector pFastBac series in the baculovirus Bac-to-Bac expression system, between Polyhedrin promotor upstream or Polyhedrin promotor and p10 promotor, select or introduce restriction enzyme site, under Polyhedrin promotor and the control of p10 promotor, be inserted in the purpose exogenous gene sequence of expressed in insect cells respectively;
(B) comprising on another vector plasmid of CMV-IE expression cassette, its two ends select respectively or introducing and step (A) in identical restriction enzyme site, make up supporting helper plasmid, in helper plasmid CMV-IE expression cassette, be inserted in the purpose exogenous gene sequence of expressing in the zooblast;
(C) CMV-IE promotor control having been inserted the helper plasmid of exogenous gene sequence or amplified fragments down carries out enzyme and cuts; reclaim the purpose fragment; be connected to through under Polyhedrin promotor and the control of p10 promotor, inserting respectively on the Bac-to-Bac expression system metastasis transplanting physique grain of exogenous gene sequence that enzyme is cut; be structured in insect cell and the zooblast all can expression alien gene the recombinant baculovirus transfer vector, be referred to as two host's transfer vectors.
(2) make up the two host Bacmid shuttle vectorss of reorganization: two host's metastasis transplanting physique grains that will comprise foreign gene transform the DH10Bac competent cell, cultivated 48 hours under 37 ℃ of conditions through homologous recombination and selectivity flat board, screening, picking pure white bacterium colony shakes the bacterium amplification, the extraction plasmid is also identified, is the two host Bacmid shuttle vectorss of reorganization.
Dual-host recombination rhabdovirus expression vector is being produced foreign protein and the application in gene therapy and nonreplication vector vaccine on the insect cell.
Obvious improvement of the present invention is that this dual-host recombination rhabdovirus expression vector includes two kinds of promoter systems, promptly contain the discernible Polyhedrin promotor of insect cell simultaneously or/and the discernible CMV-IE promotor of p10 promotor and zooblast, both can be on insect cell expression alien gene, be used to prepare functional protein, again can be on zooblast expression alien gene, as gene therapy and nonreplication vector vaccine, be a kind of multi-functional recombination rhabdovirus expression vector.Another obvious improvement of the present invention is to be used to prepare the external source functional protein as recombination rhabdovirus expression vector, in preparation external source functional protein, produce the recombinant baculovirus that can be used for gene therapy and nonreplication vector vaccine of high-gradient, promptly saved resource, reduce cost, alleviated the work workload again.
Description of drawings
Fig. 1 is the two host's transfer vector structural representations among the embodiment 1.
Fig. 2 identifies the indirect immunofluorescence figure that pig interferon γ expresses under the Polyhedrin promotor among the embodiment 1.
Fig. 3 is a shows fluorescent microscopy images of identifying egfp expression under the CMV-IE promotor among the embodiment 1.
Fig. 4 is the two host's transfer vector structural representations among the embodiment 2.
Fig. 5 is the indirect immunofluorescence figure that identifies pig interferon alpha expression under the Polyhedrin promotor among the embodiment 2.
Fig. 6 identifies the indirect immunofluorescence figure that pig interferon γ expresses under the P10 promotor among the embodiment 2.
Embodiment
Below in conjunction with specific embodiment the present invention is elaborated, but and do not limit the present invention in any way.
Embodiment 1
A kind of dual-host recombination rhabdovirus expression vector, this recombination rhabdovirus expression vector contain discernible Polyhedrin promotor of insect cell and the discernible CMV-IE promotor of zooblast simultaneously.
This dual-host recombination rhabdovirus expression vector construction process, its step is as follows:
(1) structure of dual-host recombination rhabdovirus transfer vector:
(A) based on transfer vector pFastBac1 series plasmid in the baculovirus Bac-to-Bac expression system, select the Bst1107I restriction enzyme site in Polyhedrin promotor upstream, under the control of Polyhedrin promotor, be inserted in the purpose foreign gene pig IFN-γ sequence of expressed in insect cells, the foreign gene front end that inserts can be introduced littin signal peptide (honeybee melittin signalpeptide, the HBM) gene that can reach secreting, expressing.
(a) two primers of design, pcr amplification littin signal peptide encoding gene:
P1：5′-gatcgaattcatgaaattcttagtcaacgttgcccttgtttttatggtcg-3′
P2：5′-atccgcatagatgtaagaaatgtatacgaccataaaaacaagggc-3′
P1 contains EcoR I restriction enzyme site and ATG initiator codon, and the complementation of 19 bases is arranged between primer P1 and the P2.Two primers of P1, P2 are template each other, and with Pfu DNA Polymerase amplification coding littin signal peptide gene, the expectation amplification length is 76bp, and amplified fragments is called after pig INF-γ-S respectively.Loop parameter is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 10min, totally 30 circulations; 72 ℃ are extended 10min.
(b) according to clone's pig interferon gamma gene sequences measurement result, design a pair of primer:
P3：5′-tacatctatgcggattactgccaggcgccctttttt-3′
P4：5′-agtagcatgcttagtgatggtgatggtgatgttttgatgctctctggcc-3′
P3 upstream 5 ' end and littin signal peptide encoding gene have the complementation of 15bp, and the P4 downstream has added the base sequence and the Hind III restriction enzyme site of 6 Histidines of encoding before terminator codon.With cloning vector pGEM-PoINF-γ is template, expresses fragment gene with Pfu DNA Polymerase amplification.Loop parameter is: 94 ℃ of pre-sex change 10min; 94 ℃ of sex change 45s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 30 circulations; 72 ℃ are extended 10min.Estimate that pcr amplified fragment length is 481bp, called after PoINF-γ.
(c) the littin signal coding sequence is expressed segmental PCR with PoINF-γ and is connected
Gene fragment PoINF-γ with coded signal peptide gene fragment pig INF-γ-S and coding pig interferon γ maturation protein carries out 10g/L LMP sepharose purifying and reclaims.Purified PoINF-γ-S and PoINF-γ are respectively got 1 μ L, carry out the pre-amplification of five circulations of PCR with Pfu DNA Polymerase, add primer P1 and P4 then, proceed pcr amplification, loop parameter is: 94 ℃ of pre-sex change 10min; 94 ℃ of sex change 45s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 32 circulations; 72 ℃ are extended 10min.The expectation expanding fragment length is 542bp, and front end has EcoR I restriction enzyme site, and the rear end has Hind III restriction enzyme site, called after Mels-PoIFN-γ-6His.
(d) structure of baculovirus transfer vector pFastBac I and evaluation
Amplified fragments Mels-PoIFN-γ-6His is carried out glue purification, digest with restriction enzyme EcoR I and Hind III then.Be connected on the pFastBac of same enzymic digestion I vector plasmid.Transform the JM109 competent cell, carry out the Amp+ plate screening, the picking positive colony increases, and extracts plasmid in a small amount, carries out EcoR I and Hind III enzyme is cut evaluation, and screening obtains positive metastasis transplanting physique grain pFastBac-PoIFN-γ.Carry out the exactness of sequencing with the checking insertion sequence.
(B) go out expression cassette at another carrier pAcGFP1-C1 helper plasmid that comprises the CMV-IE expression cassette by pcr amplification, and at two ends introducing Bst1107I restriction enzyme site, in helper plasmid CMV-IE expression cassette, have the green fluorescent protein indicator, in its MCS, can be inserted in the purpose exogenous gene sequence of expressing in the zooblast;
Design two primers, Bst1107 I restriction enzyme site has been designed at the primer two ends respectively, and sequence is as follows:
P5：5′-gatcgtatacttctttcctgcgttatcc-3′ P6：5′-cgtagtatactttcggcctattggttaa-3′
With the pAcGFP1-C1 plasmid is template, and pcr amplification goes out to comprise the CMV-IE promotor, the nucleotide fragments of green fluorescent protein (GFP) and multiple clone site (MCS) and SV40 polyA, and wherein GFP can be used as indicator and is used to identify expression of exogenous gene.
PAcGFP1-C1 plasmid DNA 1 μ L (100ng)
10×PCR?Buffer 5μL
2.5mM?dNTP?Mix 5μL
Primer P5 1 μ L
Primer P6 1 μ L
Ex?Taq?polymerase 1μL
Sterile?Water 36μL
Total reaction system 50 μ L
Behind the instantaneous centrifugal mixing, carry out PCR, working procedure is as follows:
95 ℃ of pre-sex change, 3min, 95 ℃ of sex change 50s, 56 ℃ of annealing 50s, 72 ℃ are extended 2min, 30 Cycles, last 72 ℃ are extended 10min.The expectation expanding fragment length is 1801bp, amplified fragments is carried out the agarose gel electrophoresis purifying reclaim, and carries out enzyme then and cuts, and system is as follows:
PCR purified product 16 μ L
10×K?Buffer 2μL
Bst1107I 2μL
Total reaction system 20 μ L
Instantaneous centrifugal mixing, 37 ℃ of enzymes are cut 3-4h.
(C) after the pcr amplified fragment enzyme that CMV-IE promotor control has been inserted the green fluorescence protein gene sequence is down cut, reclaim the purpose fragment, be connected to through under the control of Polyhedrin promotor, inserting respectively on the pFastBac I metastasis transplanting physique grain of exogenous gene sequence that enzyme of the same race is cut, be structured in insect cell and the zooblast all can expression alien gene the recombinant baculovirus transfer vector, be referred to as two host's transfer vectors, linked system is as follows:
Enzyme is cut PCR fragment purification product 5 μ L
Enzyme is cut metastasis transplanting physique grain pFastBac-PoIFN-γ 4 μ L
2×K?Buffer 10μL
T
4Dna ligase 1 μ L
Instantaneous centrifugal mixing, 16 ℃ of ligation 18h in the water-bath connecting box.
Two host's transfer vector structural representations are seen Fig. 1.
(2) two hosts structure of Bacmid shuttle vectors of recombinating: two host's metastasis transplanting physique grains that will comprise foreign gene transform the DH10Bac competent cells, through homologous recombination be coated on and contain kantlex (50 μ g.mL
-1), tetracycline (10 μ g.mL
-1), gentamicin sulphate (7 μ g.mL
-1) the selectivity flat board on, under the inducing of IPTG and X-gal, cultivated under 37 ℃ of conditions 48 hours, screening, picking pure white bacterium colony shakes the bacterium amplification, traditional alkaline lysis method of extracting plasmid performing PCR of going forward side by side is identified, is the two host Bacmid shuttle vectorss of reorganization.
(3) preparation of dual-host recombination rhabdovirus: the two host Bacmid shuttling expression plasmid vectors of the reorganization that will make up can obtain dual-host recombination rhabdovirus expression vector through the Sf9 insect cell of liposome transfection method transfection logarithmic phase.The transfection step: the Sf9 cell that will be in logarithmic phase before the transfection earlier is with Grace ' s substratum (not containing microbiotic and FBS) piping and druming cell, about 9 * 10
5Individual .mL
-1Cell evenly is positioned over six orifice plates, and the about 2ml of Grace ' s culture volume, the cell that uses this moment have cultivated 3～4d to be in semilog vegetative period, and surviving rate is greater than 97%.Allow cell attachment 1h or adherent spending the night at least.Prepare following solution at 1.5ml sterilization eppendorf pipe:
A) solution A: each transfection is added to the Grace ' s substratum of 100 μ L, mixing to the 5 μ L Bacmid that recombinates.
B) solution B: each transfection is added to the Grace ' s substratum of 100 μ L, mixing to 6 μ L CellFECTIN Reagent.
Solution B joins in the solution A mixes, at incubated at room 30～45min.In solution A and solution B mixture, add Grace ' s substratum 0.8ml, mixing; Grace ' s substratum in six orifice plates is removed in suction, with the washing of Grace ' s substratum once, adds solution A and solution B mixture then.Hatch 5h for 27 ℃.Add the insect cell perfect medium.Collecting cell and supernatant after the pathology appear in the above or cell of transfection 72h.500 * g is centrifugal, and 5min gets supernatant, cell add in a small amount supernatant inhale beat disperse freeze thawing once afterwards after, 500 * g is centrifugal, and 5min gets supernatant, after last time, supernatant mixed, by keeping in Dark Place in 4 ℃ after the 0.2 μ m filter membrane sterile filtration, be P1 for dual-host recombination rhabdovirus.P1 is for the Sf9 cell that continues to infect logarithmic phase, and results virus is P2 generation after 5 days, obtains P3 generation with quadrat method, promptly obtains the recombinant baculovirus of high-gradient, can express evaluation.
(4) indirect immunofluorescence detects the expression of dual-host recombination rhabdovirus foreign protein in insect cell: P3 is for the Sf9 insect cell suspension of recombinate shape virus infection 72h, dropping on slide glass several, 27 ℃ of absorption 1h, carefully absorb supernatant along the edge with filter paper, acetone-the ethanol (3: 2) of-20 ℃ of precoolings of cell usefulness is 5min fixedly, wash 3 times natural air drying with the PBS that contains 1%BSA; The mouse-anti pig IFN-gamma antibodies that adds dilution in 1: 2500,37 ℃ of effect 30min; Wash 3-5 time natural air drying with the PBS that contains 1%BSA; The sheep anti mouse two that adds the FITC mark of dilution in 1: 50 resists, 37 ℃ of effect 30min; Wash 3～5 times natural air drying with the PBS that contains 1%BSA; The Sf9 insect cell that fluorescent microscope is observed recombinate shape virus infection down shows the hyperfluorescence signal, its fluorescent signal mainly is distributed on the cytolemma, foreign gene pig IFN-γ under the control of proof Polyhedrin promotor has obtained expression in insect cell, and, in insect cell, realize the secretion type expression (see figure 2) owing to introduce the littin signal peptide gene.
(5) fluorescent microscope detects the expression of dual-host recombination rhabdovirus foreign protein in mammalian cell: on 6 orifice plates, ordinary method prepares the MARC-145 cell, after waiting to grow to individual layer, abandon supernatant, the P3 of inoculation preparation is for recombinant baculovirus 0.4mL, absorption 30min adds perfect medium, then at 5%CO
2, cultivate 48h for 37 ℃, abandon supernatant after, green fluorescence in the observation of cell under the inverted fluorescence microscope shows that the green fluorescence protein gene under the control of CMV-IE promotor has obtained the expression (see figure 3) in eukaryotic cell MARC-145.
Embodiment 2:
A kind of dual-host recombination rhabdovirus expression vector, this recombination rhabdovirus expression vector contain discernible Polyhedrin promotor of insect cell and p10 promotor, have the discernible CMV-IE promotor of zooblast simultaneously.
This dual-host recombination rhabdovirus expression vector construction process, its step is as follows:
(1) structure of dual-host recombination rhabdovirus transfer vector:
(A) based on transfer vector pFastBacDual plasmid in the baculovirus Bac-to-Bac expression system, under Polyhedrin promotor and the control of p10 promotor, be inserted in purpose exogenous gene sequence pig IFN-α (the Porcine interferon alpha of expressed in insect cells respectively, PoIFN-α) and IFN-γ (Porcineinterferon alpha, PoIFN-γ), simultaneously introduce littin signal peptide (honeybee melittin signal peptide, the HBM) gene order that can realize secreting, expressing respectively at its front end.
(a) design primer, pcr amplification littin signal peptide encoding gene:
P1：5′-gatcgaattcatgaaattcttagtcaacgttgcccttgtttttatggtcg-3′，
P2：5′-ctagctcgagatgaaattcttagtcaacgttgcccttgtttttatggtcg-3′，
P3：5′-atccgcatagatgtaagaaatgtatacgaccataaaaacaagggc-3′。
P1 contains EcoR I restriction enzyme site, and P2 contains Xho I restriction enzyme site, and P1 and P2 are upstream primer, and P3 is a downstream primer, and the complementation of 19 bases is arranged between the upstream and downstream primer.Two primers of P1, P3 and two primers of P2, P3 are template each other, and with Pfu DNA Polymerase amplification coding littin signal peptide gene, the expectation amplification length is 76bp, and amplified fragments is called after PoINF α-S and PoINF γ-S respectively.Loop parameter is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 10min, totally 30 circulations; 72 ℃ are extended 10min.
(b) PoINF-α and PoINF-γ express segmental pcr amplification
According to the gene sequencing result of clone pig interferon alpha, design a pair of primer:
P4：5′-tacatctatgcggattgtgacctgcctcagacccac-3′；
P5：5′-catgaagcttagtgatggtgatggtgatgctccttcttcctgagtctgtc-3′
Upstream 5 ' end and littin signal peptide encoding gene have the complementation of 15bp, and the downstream has added the base sequence and the Hind III restriction enzyme site of 6 Histidines of encoding before terminator codon.With cloning vector pGEM-PoINF α is template, expresses fragment gene with Pfu DNA Polymerase amplification, does not comprise original signal peptide sequence.Loop parameter is: 94 ℃ of pre-sex change 10min; 94 ℃ of sex change 45s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 32 circulations; 72 ℃ are extended 10min.Estimate pcr amplified fragment length 544bp, called after PoINF-α.
According to the gene sequencing result of clone pig interferon-gamma, design a pair of primer:
P6：5′-tacatctatgcggattactgccaggcgccctttttt-3′
P7：5′-agtagcatgcttagtgatggtgatggtgatgttttgatgctctctggcc-3′
Upstream 5 ' end and littin signal peptide encoding gene have the complementation of 15bp, and the downstream has added the base sequence and the Kpn I restriction enzyme site of 6 Histidines of encoding before terminator codon.With cloning vector pGEM-PoINF γ is template, expresses fragment gene with Pfu DNA Polymerase amplification.Loop parameter is: 94 ℃ of pre-sex change 10min; 94 ℃ of sex change 45s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 32 circulations; 72 ℃ are extended 10min.Estimate that pcr amplified fragment length is 481bp, called after PoINF-γ.
(c) the littin signal coding sequence is expressed segmental PCR with PoINF-α, PoINF-γ and is connected
The genetic expression fragment PoINF-α of coded signal peptide gene fragment PoINF α-S and coding pig interferon α maturation protein is carried out the recovery of 10g/L LMP sepharose purifying, purified PoINF-S1 fragment and PoINF-α are respectively got 1 μ L, carry out the pre-amplification of five circulations of PCR with Pfu DNA Polymerase, add primer P1 and P5 then, proceed pcr amplification, loop parameter is: 94 ℃ of pre-sex change 10min; 94 ℃ of sex change 45s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 32 circulations; 72 ℃ are extended 10min.The expectation expanding fragment length is 605bp, and front end has EcoR I restriction enzyme site, and the rear end has Hind III restriction enzyme site, called after Mels-PoIFN α-6His.
Equally, the genetic expression fragment PoINF-γ with coded signal peptide gene fragment PoINF γ-S and coding pig interferon γ maturation protein carries out 10g/L LMP sepharose purifying and reclaims.Purified PoINF γ-S fragment and PoINF-γ are respectively got 1 μ L, carry out the pre-amplification of five circulations of PCR with Pfu DNA Polymerase, add primer P2 and P7 then, proceed pcr amplification, loop parameter is: 94 ℃ of pre-sex change 10min; 94 ℃ of sex change 45s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 32 circulations; 72 ℃ are extended 10min.The expectation expanding fragment length is 542bp, and front end has Xho I restriction enzyme site, and the rear end has Kpn I restriction enzyme site, called after Mels-PoIFN γ-6His.
(d) structure of pFastBacDual transfer vector
To carry out glue purification through amplified fragments Mels-PoIFN α-6His that electrophoresis was identified, digest with restriction enzyme EcoR I and Hind III then.Be connected on the pFastBacDual of same enzymic digestion vector plasmid.Transform the JM109 competent cell, carry out the screening of ammonia benzyl resistant panel, picking 5-8 clone increases, and a small amount of is extracted plasmid, carries out EcoR I and Hind III enzyme is cut evaluation, screens to obtain 2 positive plasmids, called after pFastBac-Mels-PoIFN α-6His.Carry out the exactness of sequencing with the checking insertion sequence.
To carry out glue purification through amplified fragments Mels-PoIFN γ-6His that electrophoresis was identified, digest with restriction enzyme Xho I and Kpn I then.Be connected on the positive plasmid pFastBac-Mels-PoIFN α-6His of same enzymic digestion.Transform the JM109 competent cell, the screening of ammonia benzyl resistant panel, picking 5-10 clone increases, and a small amount of is extracted plasmid, carries out Xho I and Kpn I enzyme is cut evaluation, screens to obtain 3 positive plasmids.Positive plasmid carries out sequencing, with the exactness of checking insertion sequence.Called after pFastBac-Mels-PoIFN α ﹠amp; γ-6His
(B) go out expression cassette at another carrier pAcGFP1-C1 helper plasmid that comprises the CMV-IE expression cassette by pcr amplification, and at two ends introducing Bst1107I restriction enzyme site, in helper plasmid CMV-IE expression cassette, have the green fluorescent protein indicator, in its MCS, can be inserted in the purpose exogenous gene sequence of expressing in the zooblast;
Design two primers:
P8：5′-gatcgtatacttctttcctgcgttatcc-3′ P9：5′-cgtagtatactttcggcctattggttaa-3′
With the pAcGFP1-C1 plasmid is template, and pcr amplification goes out to comprise the CMV-IE promotor, the nucleotide fragments of green fluorescent protein (GFP) and multiple clone site (MCS) and SV40 polyA, and wherein GFP can be used as indicator and is used to identify expression of exogenous gene.
PAcGFP1-C1 plasmid DNA 1 μ L (100ng)
10×PCR?Buffer 5μL
2.5mM?dNTP?Mix 5μL
Primer P8 1 μ L
Primer P9 1 μ L
Ex?Taq?polymerase 1μL
Sterile?Water 36μL
Total reaction system 50 μ L
Behind the instantaneous centrifugal mixing, carry out PCR, working procedure is as follows:
95 ℃ of pre-sex change, 3min, 95 ℃ of sex change 50s, 56 ℃ of annealing 50s, 72 ℃ are extended 2min, 30 Cycles, last 72 ℃ are extended 10min.The expectation expanding fragment length is 1801bp, amplified fragments is carried out the agarose gel electrophoresis purifying reclaim, and carries out enzyme then and cuts, and system is as follows:
PCR purified product 16 μ L
10×K?Buffer 2μL
Bst1107I 2μL
Total reaction system 20 μ L
Instantaneous centrifugal mixing, 37 ℃ of enzymes are cut 3-4h.
(C) after the pcr amplified fragment enzyme that CMV-IE promotor control has been inserted the green fluorescence protein gene sequence is down cut, reclaim the purpose fragment, be connected to through under Polyhedrin promotor and the control of p10 promotor, inserting respectively on the pFastBacDual metastasis transplanting physique grain of pig IFN-α and IFN-γ that same enzyme is cut, be structured in insect cell and the zooblast all can expression alien gene the recombinant baculovirus transfer vector, be referred to as two host's transfer vectors.Linked system is as follows:
Enzyme is cut PCR fragment purification product 5 μ L
Enzyme is cut metastasis transplanting physique grain pFastBac-Mels-PoIFN α ﹠amp; γ-6His 4 μ L
2×K?Buffer 10μL
T
4Dna ligase 1 μ L
Instantaneous centrifugal mixing, 16 ℃ of ligation 18h in the water-bath connecting box.
Two host's transfer vector structural representations are seen Fig. 4.
(2) two hosts structure of Bacmid shuttle vectors of recombinating: two host's metastasis transplanting physique grains that will comprise foreign gene transform the DH10Bac competent cells, through homologous recombination be coated on and contain kantlex (50 μ g.mL
-1), tetracycline (10 μ g.mL
-1), gentamicin sulphate (7 μ g.mL
-1) the selectivity flat board on, under 37 ℃ of conditions, cultivated 48 hours, screening, picking pure white bacterium colony shakes the bacterium amplification, traditional alkaline lysis method of extracting plasmid performing PCR of going forward side by side is identified, is the two host Bacmid shuttle vectorss of reorganization.
(3) preparation of dual-host recombination rhabdovirus: the insect cell of the two host Bacmid shuttling expression plasmid vector liposome transfection method transfection logarithmic phases of the reorganization that will make up can obtain dual-host recombination rhabdovirus expression vector.The preparation method of transfection step and dual-host recombination rhabdovirus is with embodiment 1.
(4) indirect immunofluorescence detects the expression of dual-host recombination rhabdovirus foreign protein in insect cell: P3 is for the Sf9 insect cell suspension of recombinate shape virus infection 72h, dropping on two slide glasss several, 27 ℃ of absorption 1h, carefully absorb supernatant along the edge with filter paper, acetone-the ethanol (3: 2) of-20 ℃ of precoolings of cell usefulness is 5min fixedly, wash 3 times natural air drying with the PBS that contains 1%BSA; A slide glass adds the mouse-anti pig IFN-gamma antibodies that diluted into 1: 2500, and another slide glass adds the mouse-anti pig IFN-Alpha antibodies into dilution in 1: 2500,37 ℃ of effect 30min; Wash 3-5 time natural air drying with the PBS that contains 1%BSA; The sheep anti mouse two that adds the FITC mark of dilution in 1: 50 respectively resists, 37 ℃ of effect 30min; Wash 3～5 times natural air drying with the PBS that contains 1%BSA; The Sf9 insect cell that fluorescent microscope is observed recombinate shape virus infection down all shows the hyperfluorescence signal, its fluorescent signal mainly is distributed on the cytolemma, foreign gene pig IFN-α (see figure 5) and IFN-γ (see figure 6) under proof Polyhedrin promotor and the control of P10 promotor have obtained expression in insect cell, and, in insect cell, realize secretion type expression owing to all introduce the littin signal peptide gene
(5) fluorescent microscope detects the expression of dual-host recombination rhabdovirus green fluorescent protein in mammalian cell.Method is with embodiment 1.
It should be noted that at last: Application Example is set forth principle of the present invention and embodiment herein, and the explanation of above embodiment just is used for helping to understand the solution of the present invention and core concept; Simultaneously, for one of ordinary skill in the art, according to thought of the present invention, the part or more specific that all can change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention.
Claims (5)
1. dual-host recombination rhabdovirus expression vector, it is characterized in that: this recombination rhabdovirus expression vector contains discernible Polyhedrin promotor of insect cell and the discernible CMV-IE promotor of zooblast simultaneously.
2. dual-host recombination rhabdovirus expression vector according to claim 1 is characterized in that: it also contains the discernible p10 promotor of insect cell.
3. the construction process of claim 1 or 2 described dual-host recombination rhabdovirus expression vectors, it is characterized in that: this method may further comprise the steps:
(1) structure of recombinant baculovirus transfer vector:
(A) based on transfer vector pFastBac series in the baculovirus Bac-to-Bac expression system, between Polyhedrin promotor upstream or Polyhedrin promotor and p10 promotor, select or introduce restriction enzyme site, under Polyhedrin promotor and the control of p10 promotor, be inserted in the purpose exogenous gene sequence of expressed in insect cells respectively;
(B) comprising on another vector plasmid of CMV-IE expression cassette, its two ends select respectively or introducing and step (A) in identical restriction enzyme site, make up supporting helper plasmid, in helper plasmid CMV-IE expression cassette, be inserted in the purpose exogenous gene sequence of expressing in the zooblast;
(C) CMV-IE promotor control having been inserted the helper plasmid of exogenous gene sequence or amplified fragments down carries out enzyme and cuts, reclaim the purpose fragment, be connected to through under Polyhedrin promotor and the control of p10 promotor, inserting respectively on the Bac-to-Bac expression system metastasis transplanting physique grain of exogenous gene sequence that enzyme is cut, be structured in insect cell and the zooblast all can expression alien gene the recombinant baculovirus transfer vector, be referred to as two host's transfer vectors;
(2) make up the two host Bacmid shuttle vectorss of reorganization: two host's metastasis transplanting physique grains that will comprise foreign gene transform the DH10Bac competent cell, cultivated 48 hours under 37 ℃ of conditions through homologous recombination and selectivity flat board, screening, picking pure white bacterium colony shakes the bacterium amplification, the extraction plasmid is also identified, is the two host Bacmid shuttle vectorss of reorganization;
(3) preparation of recombinant baculovirus: the insect cell of the two host Bacmid shuttling expression plasmid vector transfection logarithmic phases of the reorganization that will make up can obtain dual-host recombination rhabdovirus expression vector.
4. claim 1 or 2 described dual-host recombination rhabdovirus expression vectors are produced the application in the foreign protein on insect cell.
5. claim 1 or the application of 2 described dual-host recombination rhabdovirus expression vectors in gene therapy and nonreplication vector vaccine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088063A (en) * | 2011-11-04 | 2013-05-08 | 华中农业大学 | Recombinant pseudotype baculovirus expressing rabies virus G protein and application |
| CN104711290A (en) * | 2013-12-16 | 2015-06-17 | 特菲(天津)生物医药科技有限公司 | Recombinant plasmid, recombinant baculovirus prepared from the same and application of virus in vaccine preparation |
| CN111378687A (en) * | 2018-12-27 | 2020-07-07 | 陕西杆粒生物科技有限公司 | High-yield baculovirus expression vector |
| CN111748584A (en) * | 2020-05-28 | 2020-10-09 | 东北农业大学 | Wide-host expression vector, recombinant BacMam virus prepared based on vector and application of recombinant BacMam virus |
| CN116396911A (en) * | 2023-06-01 | 2023-07-07 | 江苏聚庚科技股份有限公司 | Bacterial strain and microbial inoculum for treating pesticide wastewater and application method and device thereof |
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- 2008-10-16 CN CN200810230451A patent/CN101724651A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088063A (en) * | 2011-11-04 | 2013-05-08 | 华中农业大学 | Recombinant pseudotype baculovirus expressing rabies virus G protein and application |
| CN104711290A (en) * | 2013-12-16 | 2015-06-17 | 特菲(天津)生物医药科技有限公司 | Recombinant plasmid, recombinant baculovirus prepared from the same and application of virus in vaccine preparation |
| CN111378687A (en) * | 2018-12-27 | 2020-07-07 | 陕西杆粒生物科技有限公司 | High-yield baculovirus expression vector |
| CN111378687B (en) * | 2018-12-27 | 2023-06-23 | 陕西杆粒生物科技有限公司 | High-yield baculovirus expression vector |
| CN111748584A (en) * | 2020-05-28 | 2020-10-09 | 东北农业大学 | Wide-host expression vector, recombinant BacMam virus prepared based on vector and application of recombinant BacMam virus |
| CN116396911A (en) * | 2023-06-01 | 2023-07-07 | 江苏聚庚科技股份有限公司 | Bacterial strain and microbial inoculum for treating pesticide wastewater and application method and device thereof |
| CN116396911B (en) * | 2023-06-01 | 2023-08-04 | 江苏聚庚科技股份有限公司 | Bacterial strain and microbial inoculum for treating pesticide wastewater and application method and device thereof |
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