CN1429910A - Cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier and preparation method - Google Patents
Cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier and preparation method Download PDFInfo
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- CN1429910A CN1429910A CN 01138377 CN01138377A CN1429910A CN 1429910 A CN1429910 A CN 1429910A CN 01138377 CN01138377 CN 01138377 CN 01138377 A CN01138377 A CN 01138377A CN 1429910 A CN1429910 A CN 1429910A
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Abstract
A single embedded polyhedronvirus shuttle expression carrier of bollworm is configured through inserting the low-copy bacterium F replicon, kanamycine resistant gene and beta galactosidase gene to the polyhedrom gene site of the virus genom. The doner plasmid is used to transfer the exogenous gene to the insetion site of transposon in the said shuttle expression carrier. After bollworm cell is transfected by said DNA, the infections recombinant virus particles are formed to express exogenous gene under control of polyhedron promoter and P10 promoter.
Description
Technical field
The invention belongs to molecular biology and technical field of bioengineering, particularly relate to a kind of cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier, also relate to the preparation method of this shuttle expression carrier.
Background technology
Baculovirus is widely used in the biological control of agriculture and forestry injurious insect.The still very good eukaryotic expression system of baculovirus-insect (cell) system has been used for the research and the production of foreign gene, diagnostic reagent and vaccine etc. simultaneously.The same with other baculovirus, China cotton bollworm single particle embedded nuclear polyhedrosis virus (Helicoverpaarmigera single nucleocapsid nucleopolyhedrovirus, hereinafter to be referred as wild-type virus or HaSNPV) as sterilant owing to there is the slow-footed shortcoming of desinsection, cause the effect of its big area control bollworm harm to be restricted; And the expression amount that improves constantly virus expression systems also is the research focus of current bioengineering field always.To the molecular biological research of baculovirus, particularly will help to disclose duplicating and regularity of infection of virus, for recombinant virus sterilant and development and application quick, efficient expression vector lay the foundation to the structure of virogene and the research of function.
Utilize AcMNPV expressing human-interferon-in the Sf9 cell from nineteen eighty-three Smith and Summers reported first, rhabdovirus expression vector because of its can be in insect cell and insect larvae efficiently expressing exogenous gene, and in most of the cases baculovirus expression system has the ability of processing and modification expression product, cutting, glycosylation and phosphorylation and most of expression product as signal peptide have very high advantages such as biological activity, make it become crucial eukaryotic expression system.Use the foreign gene that baculovirus expression system has been expressed hundreds of kind different sources at present.
Traditional recombinant baculovirus makes up and need be undertaken by following two steps: at first must be on the multiple clone site of metastasis transplanting physique grain with exogenous gene cloning, and multiple clone site is positioned under the strong promoter control of baculovirus usually.With transfer vector with the baculovirus DNA cotransfection in insect cell, produce recombinant virus through homologous recombination, recombination fraction is usually between 0.1-1%.Secondly need to identify and purification of Recombinant virus with the plaque method.Because the low and plaque method of purification complicated operation, consuming time of production rate of recombinant virus often needs several months even longer time to make up a complete recombinant virus.In view of there are above-mentioned two defectives in baculovirus expression system, Kuckow has invented a kind of method that fast, effectively produces recombinant baculovirus, and promptly the Bac-to-Bac system can finish the structure of recombinant virus and efficiently expressing of foreign gene in 7 days.This system is by Bacmid and pFastBac
TMDonor plasmid
MTwo portions are formed.Bacmid contains the dna fragmentation of the 8.5kb of the mini-F replicon of a low copy number, a kalamycin resistance gene and a lacZ α gene, short-movie section as bacterium transposon insertion site (mini-attTn7) is inserted into lacZ α gene N-end, and this segmental insertion does not change the reading frame of lacZ α gene.This Bacmid can duplicate and can duplicate the complete virus particle of formation in insect cell in E.coli.PFastBac
TMDonor plasmid then is made up of a mini-Tn7 transposable element, is inserted with the expression cassette that a gentamicin resistant gene, special strong promoter of baculovirus, multiple clone site and SV40 (A) poly signal sequence are formed between the mini-Tn7 left and right arms.Under the effect of the translocase that helper plasmid provides, clone in pFastBac
TMThe foreign gene of donor plasmid multiple clone site can be indexed on the mini-attTn7 site of Bacmid, by antibiotics resistance screening and blue hickie Screening and Identification reorganization Bacmid, in insect cell, carry out expression of exogenous gene from extracting the BacmidDNA transfection in the E.coli subsequently.This method has the recombination fraction height, recombinant virus screens advantages such as convenient.
Summary of the invention:
The object of the present invention is to provide the full genome shuttle expression carrier of a kind of cotton bollworm single particle embedded nuclear polyhedrosis virus, foreign gene can be inserted fast on the full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus (HZ8), only needed 7-15 days can obtain recombinant virus, and at the cell inner expression foreign gene, simultaneously under the regulation and control of P10 strong promoter, efficiently expressing exogenous gene, the Rapid identification recombinant virus.
Another object of the present invention is to provide the preparation method of this polyhedrosis virus shuttle expression carrier.
In order to achieve the above object, the present invention adopts following technical measures: the mini-Freplicon that will contain low copy number, the dna fragmentation of a kalamycin resistance gene and a lacZ α gene 8.5kb is inserted in the polyhedrosis gene in the cotton bollworm single particle embedded nuclear polyhedrosis virus genome, made up and in E.coli, to have duplicated and can in insect cell, duplicate the cotton bollworm single particle embedded nuclear polyhedrosis virus HZ8 (Ha-Bacmid) that forms complete virus particle, we have successfully made up the HapFastPhP10 plasmid on the basis of pFastDual plasmid recently, and the eGFP gene is inserted into multiple clone site under the P10 promotor control, after transposition in the DH10B recipient bacterium, the HZ8Ph+eGFP plasmid transfection of will recombinating is expressed in the HzAm1 cell, and so far we successfully make up the perfect HaBac to Bac expression system of a cover.The full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus HaBacmid, preserving number CCTCC M201048.The full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus HZ8 is transformed into-80 ℃ of preservations in the Eschrichia Coli Dh10B strain.The structure of the full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus (HZ8Bac) is to utilize the method for homologous recombination with a DNA of bacteria F replicator (mini-F replicon) that contains low copy number, a kalamycin resistance gene and a lacZ α gene, the dna fragmentation that short-movie section of inserting site (mini-attTn7) as the bacterium transposon is inserted into the 8.5kb of lacZ α gene N-end is replaced on the virus genomic polyhedrosis gene site, therefore, the full genome shuttle expression carrier of constructed cotton bollworm single particle embedded nuclear polyhedrosis virus (HZ8) is the recombinant virus that lacks polyhedrosis gene, only can produce cytopathy with this recombinant virus infection bolworm cell (HzAm1), but can not in nucleus, form inclusion body, see figure.Utilizing donor plasmid (HapFastBac) that the transposon that foreign gene inserts to the full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus (HZ8) is inserted site (mini-attTn7), behind its DNA transfection bolworm cell (HzAm1), can form and have infective recombinant virus particle, and can be under polyhedron promotor and the control of P10 promotor expression alien gene.Multiple clone site under the constructed existing P10 promotor control of donor plasmid (pFastPhP10) that different with many embedding nuclear polyhedrosis virus of autographa california shuttling expressing system (AcBacmid) is is inserted the complete polyhedrosis gene that has the polyhedron promoter sequence again.
Its advantage is after foreign gene is inserted into multiple clone site under the P10 control, through transposition the transposon that foreign gene and polyhedrosis gene are inserted in the full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus (HaBcmid) is simultaneously inserted the site, transfection is behind bolworm cell (HzAm1), can in observation of cell under the opticmicroscope, whether form inclusion body, as forming inclusion body in the cell, the proof transfection is succeedd, and can collect recombinant virus particle cells infected and can carry out the detection of exogenous gene expression level more subsequently.Therefore the formation of the expression of polyhedrosis gene and inclusion body can be used as the important identification marking of transfection success.In addition, owing to again polyhedrosis gene is inserted into viral genome, the recombinant virus that is produced is a recombinant virus that does not lack any gene, thus this recombinant virus can not only be in insect cell also can be in insect larvae expression alien gene.Another significant advantage is to be convenient to insert the toxin gene that insect is had the specificity toxic action, makes up efficiently, recombinant virus sterilant fast.
Description of drawings
Fig. 1 is the structure synoptic diagram of cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle plasmid;
Through the method for homologous recombination with bacterium DNA replication (mini-Freplicon) that contains low copy number, a kalamycin resistance gene and a lacZ α gene, the dna fragmentation that short-movie section of inserting site (mini-attTn7) as the bacterium transposon is inserted into the 8.5kb of lacZ α gene N-end is replaced on the genomic polyhedrosis gene of cotton bollworm single particle embedded nuclear polyhedrosis virus (HaSNPV) site the full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus (HaBacmid) that structure can duplicate and can duplicate in bacterium in insect cell or insect body.
Fig. 2 is the expression synoptic diagram of foreign gene at the cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression system;
The schema of expression alien gene in the bolworm cell (HzAm1) is arrived in foreign gene transfection subsequently in donor plasmid (HapFastPhP10) imports the full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus (HaBacmid).
Fig. 3 is the pathological section synoptic diagram of bollworm simple grain embedding caryogram polyhedron shuttle expression carrier DNA transfection bolworm cell.
The full genome shuttle expression carrier of cotton bollworm single particle embedded nuclear polyhedrosis virus (HaBacmid) infects the pathological section of bolworm cell (HzAm1).
Embodiment
Its step is as follows:
1, contains the activation of the DH10B recipient bacterium 1 of HZ8 (HaBacmid) and helper plasmid.To be stored in-80 ℃ of DH10B recipient bacteriums is inoculated on the LB solid medium that contains kantlex (50 μ g/ml) and tsiklomitsin (50 μ g/ml), 37 ℃, cultivated 12-16 hour, selecting single colony inoculation contains on the LB liquid nutrient medium of kantlex (50 μ g/ml) and tsiklomitsin (50 μ g/ml) to 3mL, cultivated 24 hours, and prepared the competence recipient bacterium according to a conventional method for 37 ℃.
2, contain the activation of DH5 α bacterium of HapFastPhP10 plasmid and the extraction of HapFastPhP10 plasmid 2.To be stored in-80 ℃ of DH5 α recipient bacteriums is inoculated on the LB solid medium that contains gentamicin (50 μ g/ml), 37 ℃, cultivated 12-16 hour, selecting single colony inoculation contains on the LB liquid nutrient medium of gentamicin (50 μ g/ml) to 3mL, cultivated 16 hours for 37 ℃, extract the method for plasmid routinely and extract the HapFastPhP10 plasmid.
3, foreign gene is connected in the multiple clone site of HapFASTPhP10 plasmid 3, subsequently with recombinant plasmid transformed in the DH10B recipient bacterium that contains HZ8 (HaBacmid) and helper plasmid 4, with recipient bacterium be placed in 1 milliliter of LB liquid nutrient medium that contains tsiklomitsin, kalamycin, gentamicin 37 ℃ cultivate 4 hours after, get 100 microlitre thalline and be inoculated on the solid plate substratum that contains above-mentioned three kinds of antibiotic X-, cultivated 24 hours for 37 ℃.Selecting white bacterial classification inoculation contains in above-mentioned three kinds of antibiotic LB liquid nutrient mediums 37 ℃ in 3 milliliters and cultivated 24 hours.Extract plasmid HZ8 (HaBacmid) DNA that is inserted with foreign gene according to a conventional method.
4, according to the transfection method of routine, utilize plasmid HZ8 (HaBacmid) the DNA transfection that liposome will contain foreign gene to cultivate 4-7 days for interior 28 ℃ to the HzAm1 cell, whether examine under a microscope has inclusion body to occur 5 in the cell.
5, utilize ordinary method to detect expression of exogenous gene amount 6.
Claims (2)
1, a kind of cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier, HZ8, HaBacmid, CCTCC M 201048.
2, a kind of preparation method who realizes the described cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier of claim 1 comprises the following steps:
A, contain the activation of the DH10B recipient bacterium (1) of HaBacmid and helper plasmid, be inoculated on the LB solid medium that contains kantlex and tsiklomitsin being stored in-80 ℃ of DH10B recipient bacteriums, cultivated 12-16 hour for 37 ℃, selecting single colony inoculation contains on the LB liquid nutrient medium of kantlex and tsiklomitsin to 3mL, cultivated 24 hours, and prepared the competence recipient bacterium according to a conventional method for 37 ℃;
B, the activation of DH5 α bacterium that contains the pFastPhP10 plasmid and the extraction of pFastPhP10 plasmid (2), be inoculated on the LB solid medium that contains gentamicin being stored in-80 ℃ of DH5 α recipient bacteriums, 37 cultivated 12-16 hour, selecting single colony inoculation contains on the LB liquid nutrient medium of gentamicin to 3mL, cultivated 16 hours for 37 ℃, extract the method for plasmid routinely and extract the HapFastPhP10 plasmid;
C, foreign gene is connected in the multiple clone site of pFastPhP10 plasmid (3), subsequently with recombinant plasmid transformed (4) in the DH10B recipient bacterium that contains HaBacmid and helper plasmid, recipient bacterium being placed in the LB liquid nutrient medium that contains tsiklomitsin, kalamycin, gentamicin 37 ℃ cultivated after 4 hours, get 100 microlitre thalline and be inoculated on the solid plate substratum that contains above-mentioned three kinds of antibiotic X-, cultivated 24 hours for 37 ℃.Select white bacterial classification inoculation in containing above-mentioned three kinds of antibiotic LB liquid nutrient mediums 37 ℃ cultivated 24 hours, extract the plasmid HaBacmid DNA that is inserted with foreign gene according to a conventional method;
The transfection method of D, more solito utilizes the plasmid HaBacmid DNA transfection that liposome will contain foreign gene to cultivate 4-7 days for interior 28 ℃ to the HzAm1 cell, and whether examine under a microscope has inclusion body (5) to occur in the cell.
E, utilize ordinary method to detect expression of exogenous gene amount (6).
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352371A (en) * | 2011-10-14 | 2012-02-15 | 苏州金唯智生物科技有限公司 | PC series plasmid as well as construction method and application thereof |
| CN103898068A (en) * | 2012-12-26 | 2014-07-02 | 中国科学院遗传与发育生物学研究所 | Recombinant baculovirus expressing insect prothoracicotropic hormone gene, and its application |
| CN103898111A (en) * | 2012-12-26 | 2014-07-02 | 中国科学院遗传与发育生物学研究所 | Recombinant baculovirus expressing dsRNA inhibiting insect juvenile hormone binding protein gene |
| CN111808884A (en) * | 2020-07-23 | 2020-10-23 | 云舟生物科技（广州）有限公司 | Baculovirus expression system and construction method and application thereof |
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2001
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352371A (en) * | 2011-10-14 | 2012-02-15 | 苏州金唯智生物科技有限公司 | PC series plasmid as well as construction method and application thereof |
| CN102352371B (en) * | 2011-10-14 | 2013-05-01 | 苏州金唯智生物科技有限公司 | PC series plasmid as well as construction method and application thereof |
| CN103898068A (en) * | 2012-12-26 | 2014-07-02 | 中国科学院遗传与发育生物学研究所 | Recombinant baculovirus expressing insect prothoracicotropic hormone gene, and its application |
| CN103898111A (en) * | 2012-12-26 | 2014-07-02 | 中国科学院遗传与发育生物学研究所 | Recombinant baculovirus expressing dsRNA inhibiting insect juvenile hormone binding protein gene |
| CN103898068B (en) * | 2012-12-26 | 2016-03-30 | 中国科学院遗传与发育生物学研究所 | Express recombinant baculovirus and the application thereof of insect prothoracic gland hormone gene |
| CN103898111B (en) * | 2012-12-26 | 2016-12-28 | 中国科学院遗传与发育生物学研究所 | The recombinant baculovirus of the dsRNA of expression inhibiting corpus allatum hormone binding-protein gene |
| CN111808884A (en) * | 2020-07-23 | 2020-10-23 | 云舟生物科技（广州）有限公司 | Baculovirus expression system and construction method and application thereof |
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