CN101481702B - Recombinant vector containing polyhedrosis gene, and method for expressing and purifying protein - Google Patents
Recombinant vector containing polyhedrosis gene, and method for expressing and purifying protein Download PDFInfo
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Abstract
The invention relates to the technical field of gene engineering, in particular to a recombinant vector containing polyhedrosis genes and a method for expressing and purifying proteins. The invention provides a recombinant plasmid pBacPAKph, a recombinant plasmid pBacPAKph-M and a linear virus recon with a connection number of CGMCC No.2768. Linear virus recon BmBacPAKph-M is adopted to infect a host body and express fusion protein of polyhedrosis protein and target protein, the expressed fusion protein is separated and purified, enzyme digestion is carried out on prolease to obtain the target protein. The purified target protein can be prepared into intravenous medicines or used as an object for study on protein functions. A plurality of proteins which are provided by the invention and hard to be expressed and purified but can be easily expressed and purified by protein expression and purification systems, especially membrane proteins have a broad prospect of application.
Description
Technical field
The present invention relates to gene engineering technology field, relate in particular to the method for a kind of recombinant plasmid that contains polyhedron gene and preparation method thereof and protein expression, purifying.
Background technology
Insect cell expression system (being baculovirus expression system) has unique biological characteristics, day by day is subject to people's attention.The gene of baculovirus is in utmost point late gene expression process, the albumen that two kinds of high efficient expressions are arranged, they are polyhedrin and P10 albumen: polyhedrin is the main component that forms inclusion body, infecting the accumulation of later stage in cell can be up to 30%～50%, forming xln, is the nonessential composition of virus replication; P10 albumen also is that a viroid is copied nonessential composition, can form fibrous material in cell.Polyhedrosis gene and P10 gene all are positioned now and clone, and the promotor of these two genes has stronger startup ability, so these two gene locuss become the desirable foreign gene insertion point of rhabdovirus expression vector.
Because the Baculovirus Gene group is very large, be not suitable for using conventional plasmid clone technology, but goal gene is cloned in the transfer vector, by homologous recombination, goal gene is transferred in the baculovirus again.Linearizing virus is more conducive to restructuring than ring-shaped DNA molecule, therefore, people have made up BacPAK6 virus, it has three Bsu36I restriction enzyme sites, virus produces two small segments and a viral DNA large fragment after cutting through enzyme, and this not only makes viral linearizing, also make the polyhedron gene fragment that is positioned at polyhedrin promotor downstream cut, make simultaneously the indispensable gene ORF1629 inactivation of virus replication.So only have after recombinating successfully, indispensable gene is repaired, linearizing virus is just brought back to life.BmBacPAK6 virus and BacPAK6 virus type seemingly have baculovirus polyhedrin body strong promoter and three Bsu36I restriction enzyme sites, but the infected silkworm cell that it can be single-minded.It is recombinated in the BmN bombyx mori cell by BacPAK6 DNA and wild-type silkworm baculovirus BmNPV DNA and realizes.BmBacPAK6 forms linearizing virus after the Bsu36I enzyme is cut, the disappearance polyhedron gene.
When utilizing traditional recombinant insect virus screening with the foreign gene recombinant virus, need to become the form of plaque to screen with microscope by virus infected cell, except the needs masterful technique, also time-consuming, cycle is long, the restructuring target protein that obtains is less, is unfavorable for preservation, and the purifying of target protein is also relatively more difficult.In addition, membranin is difficult for purifying because of himself hydrophobic property, and when expressing in the expression systems such as intestinal bacteria, yeast cell, membranin generally all can be incorporated in the cytolemma, has greatly increased the difficulty of purifying.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of recombinant plasmid pBacPAKph that contains polyhedron gene and preparation method thereof.
Technical problem to be solved by this invention also is to provide a kind of recombinant plasmid pBacPAKph-M that contains polyhedron gene and target protein fusion gene.
It is the method for linearizing virus recon of CGMCC No.2768 and preparation method thereof, screening method and matter expression and purification albumen that technical problem to be solved by this invention also is to provide a kind of preserving number.
Technical scheme of the present invention is as follows:
Technical problem to be solved by this invention is achieved in that
The present invention at first provides a kind of recombinant plasmid pBacPAKph, for the EcoR V of pBacPAK8 plasmid and BamH I double digestion small segment by one section recombinant plasmid that nucleotide sequence substituted, described nucleotide sequence is connected in sequence by the suddenlyd change polyhedrin encoder block sequence of deletion and the nucleotide sequence of coding TEV proteolytic enzyme restriction enzyme site of polyhedron nucleic acid sequence of promoter, terminator.Made by the preparation method who may further comprise the steps:
(1) take silkworm baculovirus DNA as template, sequence is primer shown in SEQ ID No.1 and the SEQ ID No.2, by pcr amplification reaction, makes amplified production;
(2) the amplified production EcoR V and the BamH I double digestion that step (1) are made are cloned into the pBacPAK8 plasmid, make recombinant plasmid pBacPAKph.
Those skilled in the art according to general knowledge as can be known, TEV proteolytic enzyme described herein also can realize that proteolytic enzyme of the present invention substitutes with other.
The present invention also provides a kind of recombinant plasmid pBacPAKph-M, is inserted with the recombinant plasmid of target protein gene M for the multiple clone site of recombinant plasmid pBacPAKph;
(this linearizing virus recon BmBacPAKph-M is by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation to have the present invention further provides a kind of preserving number and be the linearizing virus recon of CGMCC No.2768, the address is in Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date is on November 28th, 2008, deposit number is CGMCC No.2768, the Classification And Nomenclature of biomaterial: Bombyx mori nuclear polyhydrosis virus Bombyx mori nucleopolyhedrovirus).
Above-mentioned preserving number is that the preparation method of the linearizing virus recon of CGMCC No.2768 is: made by recombinant plasmid pBacPAKph-M and linearizing BmBacPAK6 viral DNA homologous recombination.
Above-mentioned preserving number is the screening method of the linearizing virus recon of CGMCC No.2768: utilize polyhedrin to screen the virus that homologous recombination successfully occurs, by having or not polyhedron to bring back to life and whether Show Color is as selection markers, obtain the linearizing restructuring with the polyhedrosis gene function and realize the virus of successful generation homologous recombination of the high efficient expression of fusion rotein.
Above-mentioned preserving number is the linearizing virus recon expression and purification method of protein of CGMCC No.2768, may further comprise the steps:
(1) utilizes preserving number to be the linearizing virus recon infection host body of CGMCC No.2768, express the fusion rotein of polyhedrin and target protein;
(2) fusion rotein of separating step (1) expression, purifying, proteolytic enzyme enzyme are cut and are obtained target protein.
Above-mentioned preserving number is that the separation of fusion rotein utilizes polyhedrin to be easy to form the albumen crystallization and realizes in the expression and purification method of protein of linearizing virus recon of CGMCC No.2768; The purifying of target protein utilizes the polyhedrin antibody affinity chromatography and the fusion rotein behind the purifying is carried out the proteolytic enzyme enzyme cut and realize.
The technique effect that the present invention realizes is as follows:
Polyhedrosis gene and goal gene are merged in the present invention, be cloned in the transfer vector, by with linearizing BmBacPAK6 virus homologous recombination occurs in the silkworm cells BmN, the recon that obtains has not only recovered indispensable gene ORF1629 again, bring back to life virus, and recovered polyhedrosis gene.Thereby, when recombinant virus screens, except according to traditional method, also can be according to having or not polyhedron to judge recombinant virus.This new expression system can obtain the target protein of more recombinating, and target protein can come purifying by polyhedron, cuts polyhedrin removed by the proteolytic enzyme enzyme again to obtain pure target protein.This new expression system also can be used for expressing, some difficulties of purifying are expressed, the albumen of purifying, such as membranin.Membranin is difficult for purifying because of himself hydrophobic property, and when expressing in the expression systems such as intestinal bacteria, yeast cell, membranin generally all can be incorporated in the cytolemma, greatly increased the difficulty of purifying, and the present invention can solve this problem.During amount reproduction, its host cell does not copy baculovirus in cell, and membranin and polyhedrin merge, so membranin is difficult for being incorporated in the host cell membrane.
Utilize the recombinant plasmid that contains polyhedron gene provided by the invention can effectively express and the various target proteins of purifying, membranin particularly, be with a wide range of applications, for solving difficult express and the obtaining of albumen of purifying is extremely important in the prior art.
Description of drawings
Fig. 1 is the vector construction schematic diagram that the specific embodiment of the invention provides.
Among the figure, pPh: polyhedron promotor; Ph: polyhedrosis gene; TEV:TEV proteolytic enzyme restriction enzyme site zone.
Fig. 2 is the plasmid map schematic diagram of the recombinant plasmid pBacPAKph (being recombinant transfer vector pBacPAKph) that provides of the specific embodiment of the invention.
Among the figure, the deletion that suddenlyd change of the terminator codon of polyhedrosis gene polyhedrin, the downstream adds multiple clone site MCS, is convenient to the insertion of external source goal gene.
Fig. 3 is the expression schematic diagram of the fusion rotein that provides of the specific embodiment of the invention.
Among the figure, the polyhedron fusion rotein of band shown in the arrow for expressing, size is about 71kD, and fusion rotein obtains great expression.
Fig. 4 is the target protein schematic diagram behind the purifying that provides of the specific embodiment of the invention
Among the figure, M: standard protein molecular weight; 1: the target protein band behind the purifying of 42kDa place existence.
Biological preservation explanation:
The Classification And Nomenclature of biomaterial: Bombyx mori nuclear polyhydrosis virus
Latin name: Bombyx mori nucleopolyhedro virus
Depositary institution's full name: China Committee for Culture Collection of Microorganisms common micro-organisms center
Deposit number: CGMCC No.2768
Preservation date: on November 28th, 2008
Depositary institution address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica
Embodiment
The technology used in the present invention means, those skilled in the art all can be according to " molecular cloning " or other technologies handbooks and are known.
1. design of primers
Amplification polyhedron promotor and polyhedrosis gene are introduced proteolytic enzyme TEV restriction enzyme site, as shown in Figure 1 simultaneously.Among the figure, pPh: polyhedron promotor; Ph: polyhedrosis gene; TEV:TEV proteolytic enzyme restriction enzyme site zone.
Design of primers is as follows:
Upstream primer F:CAAGCT
ACTGTCGACAAGCTCTGTCC (italic represents EcoR V restriction enzyme site) SEQ ID No.1;
Downstream primer R:AGCTAC
ATACGCCGGACCAGTGAACA (italic represents BamH I restriction enzyme site, and runic is TEV proteolytic enzyme restriction enzyme site) SEQ ID No.2.
Polyhedrosis gene promotor pPh downstream keeps polyhedrosis gene coding region Ph, simultaneously the terminator codon sudden change of coding region Ph is deleted, add the restriction enzyme site zone of proteolytic enzyme TEV behind the Ph, nucleotide sequence is GAAAACCTGTATTTTCAGGGC, and it is Glu-Asn-Leu-Tyr-Phe-Gln that coded corresponding aminoacid sequence and proteolytic enzyme enzyme are cut the position
Gly.
2.PCR amplification
Take silkworm baculovirus DNA as template, the PCR reaction parameter is designed to: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 1min, 68 ℃ of renaturation are extended altogether 2min, 30 circulations, 72 ℃ are extended 5min.
In the centrifuge tube of 0.5ml, add following component:
10×PCR Buffer 10μL；
25mmol MgCl
2 8μL；
2.5mmol dNTPs 8μL；
F 1μL；
R 1μL；
Template 1 μ L;
KOD-Plus archaeal dna polymerase (TOYOBO company, Japan) 1 μ L;
Add aseptic double-distilled water to 100 μ L.
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified the amplification segment, cut simultaneously glue and reclaimed the purpose segment.
3. make up recombinant transfer vector pBacPAKph
The PCR product through the gene fragment clone behind EcoR V and the BamH I double digestion in the pBacPAK8 of EcoR V and BamH I double digestion (available from Clontech company), the gene of pcr amplification is connected on the carrier, be built into recombinant transfer vector pBacPAKph, its plasmid map as shown in Figure 2, through enzyme cut, sequencing analysis, identify that recombination sequence is correct.The terminator codon of the polyhedrosis gene polyhedrin deletion that suddenlyd change among the recombinant transfer vector pBacPAKph.The multiple clone site MCS that utilizes the downstream to add inserts the external source goal gene.Gained recombinant transfer vector pBacPAKph is recombinant plasmid pBacPAKph.
4. the structure of recombinant transfer plasmid pBacPAKph-M
Among the recombinant transfer vector pBacPAKph, there is a multiple clone site (MCS) in the downstream of polyhedron (polyhedrin) gene, can inserts any foreign gene.Among the present invention take the silkworm membranin as example.
Upstream primer F2:CTAC
ATGTCGATATCTATGGACGC (italic is the BamHI site);
Downstream primer R2:CTAAGA
TTATGCTGTCGGTTTCTCTTCC (EcoR I).
Take silkworm cDNA as template, the PCR reaction parameter is designed to: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 1min, 65 ℃ of renaturation are extended 2min, 30 circulations, 72 ℃ are extended 5min.
In the centrifuge tube of 0.5ml, add following component:
10×PCR Buffer 10μL；
25mmol MgCl
2 8μL；
2.5mmol dNTPs 8μL；
F2 1μL；
R2 1μL；
Template 1 μ L;
KOD-Plus archaeal dna polymerase (TOYOBO company, Japan) 1 μ L;
Add aseptic double-distilled water to 100 μ L.
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified the amplification segment, cut simultaneously glue and reclaimed the purpose segment.
The PCR product in the recombinant transfer vector pBacPAKph of BamH I and EcoR I double digestion, is built into recombinant transfer plasmid pBacPAKph-M through the gene fragment clone behind BamH I and the EcoR I double digestion.Through enzyme cut, sequencing analysis, identify that recombination sequence is correct.Gained recombinant transfer plasmid pBacPAKph-M is recombinant plasmid pBacPAKph-M.
The used silkworm membranin of the present invention (note is M) is the expressed albumen of gene order of NM_001043417 for accession number among the GenBank, and the theoretical molecular size is 42.2kDa.
5. the acquisition of silkworm with recombinant baculovirus BmBacPAKph-M
Get 5 μ L recombinant transfer plasmid pBacPAKph-M DNA and 6 μ L through the modification C-type virus C BmBacPAK6 of Bsu36I linearization for enzyme restriction DNA (available from the biochemical cell in Shanghai institute), the TC-100 substratum (GIBCOBRL company) that adds 100 μ L serum-frees is mixed.The TC-100 substratum of getting 6 μ L Dosper (Bao Ling Man) adding, 100 μ L serum-frees is mixed.The BmN cell (available from the biochemical cell in Shanghai institute) of cultivating in advance in the ware of 35mm plane is used the TC-100 substratum washed twice of serum-free, and dropwise add pBacPAKph-M transferring plasmid and Dosper mixture, cultivated 4-5 days, and collected supernatant liquor for 27 ℃.Get the BmN cell in the ware of 5 μ L supernatant liquors infection 35mm plane, abandoning supernatant adds TC-100 substratum and the low melting-point agarose of balanced mix after 1 hour.After 4-5 days, form in the crystallization of optical microphotograph Microscopic observation polyhedron, if observe the polyhedron crystallization, then this polyhedrosis virus is required recombinant baculovirus BmBacPAKph-M.Gained recombinant baculovirus BmBacPAKph-M is the linearizing virus recon that preserving number is CGMCC No.2768.This linearizing virus recon BmBacPAKph-M is by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, the address is in Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date is on November 28th, 2008, deposit number is CGMCC No.2768, the Classification And Nomenclature of biomaterial: Bombyx mori nuclear polyhydrosis virus Bombyx mori nucleopolyhedrovirus.
6. the collection of linearizing virus recon BmBacPAKph-M and the expression of fusion rotein
Recombinant baculovirus BmBacPAKph-M puts into 28 ℃ of thermostat containers and cultivates with 10MOI dosage infected silkworm larva.After 5-6 days, collect larva, its single head is collected in the clean 1.5mL centrifuge tube.Worm corpse in the centrifuge tube is shaken at a high speed, stir with toothpick in case of necessity, add 1mL 1 * PBS, the abundant mixing of 10 μ L10%SDS; The centrifugal 5min of 500rpm gets supernatant liquor; Change supernatant liquor over to another centrifuge tube, the centrifugal 10min of 4000rpm gets precipitation, and is resuspended with 1mL 1 * PBS (pH=8.0).4000rpm is centrifugal again, repeatedly, until polyhedrosis virus is purer.Add 1mL 1 * PBS resuspended in the precipitation of gained, get 20 μ L and run 10%SDS-PAGE, the result as shown in Figure 3.Among the figure, the polyhedron fusion rotein of band shown in the arrow for expressing, a large amount of protein expressions appear in size about 71kD, be the fusion rotein of desired acquisition.
7. the purifying of target protein
The fusion rotein that obtains is used the affinity chromatography column purification of coupling polyhedrin antibody, obtain pure fusion rotein, again it is carried out TEV proteolytic enzyme enzyme and cut, enzyme is cut product and is passed through coupling polyhedrin antibody affinity column purifying again, finally obtains pure purpose membranin.
At first, from polyhedrosis virus, extract the polyhedron fusion rotein, obtain pure polyhedron fusion rotein by coupling polyhedrin antibody affinity column purifying.Then, in the 1.5ml centrifuge tube, mix following component: polyhedron fusion rotein 40 μ g, 0.1M DTT 3 μ L, 20XrTEV Buffer 15 μ L, rTEV proteolytic enzyme (HaiGene company) 2 μ L mend pure water to 300 μ L again.Hatch more than the 30min for 30 ℃.The centrifugal 10min of 4000rpm collects supernatant liquor after coupling polyhedrin antibody affinity column, collects effluent liquid and is the target protein M that removes rTEV proteolytic enzyme.Carry out again affinity chromatography one time, can obtain pure target protein M.Electrophoresis result as shown in Figure 4.Among the figure, M: standard protein molecular weight; 1: be presented at the 42kDa place and have a large amount of albumen, be the target protein band behind the purifying.
The listed embodiment of the present invention only is used for being convenient to understand essence of the present invention, is not used in the scope of the present invention that limits.The change that does not break away from essence of the present invention that those skilled in the art carry out the present invention according to general knowledge still belongs to protection scope of the present invention.
Claims (8)
1. recombinant plasmid pBacPAKph is characterized in that: described recombinant plasmid is the pBacPAK8 plasmid
EcoR V and
BamH I double digestion small segment is by one section recombinant plasmid that nucleotide sequence substituted, and described nucleotide sequence is connected in sequence by the suddenlyd change polyhedrin encoder block sequence of deletion and the nucleotide sequence of coding TEV proteolytic enzyme restriction enzyme site of polyhedron nucleic acid sequence of promoter, terminator;
Make by following steps:
(1) take silkworm baculovirus DNA as template, sequence is primer shown in SEQ ID No.1 and the SEQ ID No.2, by pcr amplification reaction, makes amplified production;
(2) amplified production that step (1) is made
EcoR V and
BamH I double digestion is cloned into the pBacPAK8 plasmid, makes recombinant plasmid pBacPAKph.
2. recombinant plasmid pBacPAKph-M, it is characterized in that: described recombinant plasmid pBacPAKph-M is the recombinant plasmid that the multiple clone site of the described recombinant plasmid pBacPAKph of claim 1 is inserted with the target protein gene M, and wherein said target protein gene M is the silkworm membrane protein gene.
3. the preserving number linearizing virus recon that is CGMCC No. 2768.
4. the described preserving number of claim 3 is the preparation method of the linearizing virus recon of CGMCC No. 2768, it is characterized in that: made by recombinant plasmid pBacPAKph-M claimed in claim 3 and linearizing BmBacPAK6 viral DNA homologous recombination.
5. the described preserving number of claim 3 is the screening method of the linearizing virus recon of CGMCC No. 2768, it is characterized in that: utilize polyhedrin to screen the virus that homologous recombination successfully occurs, by having or not polyhedron to bring back to life and whether Show Color is as selection markers, obtain the linearizing restructuring with the polyhedrosis gene function and realize the virus of successful generation homologous recombination of the high efficient expression of fusion rotein.
6. the described preserving number of claim 3 is the linearizing virus recon expression and purification method of protein of CGMCC No. 2768, it is characterized in that:
(1) utilizes preserving number claimed in claim 3 to be the linearizing virus recon infection host body of CGMCC No. 2768, express the fusion rotein of polyhedrin and target protein;
(2) fusion rotein of separating step (1) expression, purifying, proteolytic enzyme enzyme are cut and are obtained target protein.
7. preserving number according to claim 6 is the linearizing virus recon expression and purification method of protein of CGMCC No. 2768, and it is characterized in that: the separation of described fusion rotein utilizes polyhedrin to be easy to form the albumen crystallization and realizes.
8. preserving number according to claim 6 is the linearizing virus recon expression and purification method of protein of CGMCC No. 2768, it is characterized in that: the purifying of target protein utilizes the polyhedrin antibody affinity chromatography and the fusion rotein behind the purifying is carried out the proteolytic enzyme enzyme cut and realize.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910006011 CN101481702B (en) | 2009-01-22 | 2009-01-22 | Recombinant vector containing polyhedrosis gene, and method for expressing and purifying protein |
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| CN102516369A (en) * | 2011-12-21 | 2012-06-27 | 天津耀宇生物技术有限公司 | Mutant polyhedron and preparation method thereof |
| CN102517319A (en) * | 2011-12-27 | 2012-06-27 | 天津耀宇生物技术有限公司 | Method for purifying fusion protein based on bombyx mori baculovirus polyhedron dissolving characteristic |
| CN102952807B (en) * | 2012-11-13 | 2015-07-08 | 天津耀宇生物技术有限公司 | Pre-180 bp segments of polynedron gene and application thereof |
| CN103834676B (en) * | 2014-02-28 | 2016-12-07 | 中国科学院福建物质结构研究所 | A kind of E. coli secretion expresses plasmid vector and the construction method thereof of foreign protein |
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| CN1162017A (en) * | 1996-04-08 | 1997-10-15 | 中国农业科学院上海家畜寄生虫病研究所 | Systematic production of recombined Nippon blood-flukes glutathione transferase by using domestic silk-worm |
| CN1242428A (en) * | 1998-07-17 | 2000-01-26 | 中国科学院上海生物化学研究所 | Gene engineering carrier for recombination salvation linearization silk worm nuclear polyhedral virus |
| CN1365390A (en) * | 1999-07-27 | 2002-08-21 | 数位生物技术株式会社 | A human lactoferrin produced by using insect cell and method using the same |
| CN1405310A (en) * | 2001-08-15 | 2003-03-26 | 浙江中奇生物药业股份有限公司 | Method for producing medicine using silkworm expressed numan epidermal growth factor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1162017A (en) * | 1996-04-08 | 1997-10-15 | 中国农业科学院上海家畜寄生虫病研究所 | Systematic production of recombined Nippon blood-flukes glutathione transferase by using domestic silk-worm |
| CN1242428A (en) * | 1998-07-17 | 2000-01-26 | 中国科学院上海生物化学研究所 | Gene engineering carrier for recombination salvation linearization silk worm nuclear polyhedral virus |
| CN1365390A (en) * | 1999-07-27 | 2002-08-21 | 数位生物技术株式会社 | A human lactoferrin produced by using insect cell and method using the same |
| CN1405310A (en) * | 2001-08-15 | 2003-03-26 | 浙江中奇生物药业股份有限公司 | Method for producing medicine using silkworm expressed numan epidermal growth factor |
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