CN101429524A - Viral vectors for improving carrier transduction and gene expression efficiency, and uses thereof - Google Patents
Viral vectors for improving carrier transduction and gene expression efficiency, and uses thereof Download PDFInfo
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- CN101429524A CN101429524A CNA2008102072066A CN200810207206A CN101429524A CN 101429524 A CN101429524 A CN 101429524A CN A2008102072066 A CNA2008102072066 A CN A2008102072066A CN 200810207206 A CN200810207206 A CN 200810207206A CN 101429524 A CN101429524 A CN 101429524A
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a viral vector capable of improving the efficiency of transduction and expression of exogenous genes in animal cells and an application method thereof. The invention provides a viral vector capable of improving the efficiency of vector transduction and gene expression, which comprises a nucleus capsid protein promoter sequence, a nucleus capsid protein coded sequence and an objective gene sequence with promoter of virus, and the vector also comprises a cell transduction peptide coded sequence; and the nucleus capsid protein promoter controls transcription and translation of cell transduction peptide. The cell transduction efficiency of the modified viral vector is doubled compared with the virus, and the expression efficiency of the objective gene is also obviously improved. The invention is suitable to produce protein with natural activity, and utilizes the viral vector to perform the disease gene treatment and the vaccine immunity.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to that a kind of raising is transduceed and the virus vector and the application method thereof of expression alien gene efficient in zooblast.
Background technology
Baculovirus is the virus that a class natural host of nature existence is mainly lepidopteran and coleopteron, is used as biological pesticide for a long time and prevents and treats agriculture and forestry injurious insect, also is widely used in and produces proteinic gene engineering expression carrier in the insect cell.Its type species are autographa california nuclear polyhedrosis virus (Autographa californica multicapsid nucleopolyhedrovirus is called for short AcMNPV).
In recent years, it is found that baculovirus is the good proteinic gene engineering expression carrier of production and gene therapy and vaccine carrier in mammalian cell of a class equally.Present verified this carrier mammalian cell in tens of kinds of all kinds of sources of can transduceing effectively, and allow goal gene in mammalian cell, express (Cheng T efficiently, Xu CY, Wang YB, Chen M, Wu T, Zhang J, Xia NS.2004.A rapid and efficient method to express target genes in mammalian cells bybaculovirus, World J Gastroenterol.10 (11): 1612-1618; Hu YC 2006.Baculovirus vectors for gene therapy.Adv Virus Res.; 68:287-320.).
The ultimate principle that baculovirus vector mediation goal gene is expressed in mammalian cell is: utilization can be controlled goal gene by the promotor---as the IE promotor (CMV) of cytomegalovirus and LTR promotor of Lloyd's's tumor virus etc.---that mammalian cell is discerned, and constitutes expression cassette.DNA with this expression cassette is incorporated on the viral DNA skeleton again, makes up to obtain recombinant baculovirus; The characteristics of utilizing recombinant baculovirus can enter multiple mammalian cell are transduceed goal gene into purpose clone and are expressed.The major advantage of baculovirus-mammalian cell expression vector comprises: viral genetic background is clear, and the gene capacity is big; Can carry bigger goal gene; Biological safety is good, does not infect the mankind under the natural situation, does not duplicate in mammalian cell; The expansion scale reaches production requirement easily; Multiple mammalian cell or the like (Hu YC 2006.Baculovirus vectors for gene therapy.Adv VirusRes. can transduce; 68:287-320.; Kost T.A., Condreay J.P.and Jarvis D.L., 2005.Baculovirus asversatile vectors for protein expression in insect and mammalian cells.Nat.Biotechnol.23,567-575).But the shortcoming of this carrier system is also apparent in view, as: to the gene transfering efficiency difference of different cells, especially in genetically engineered cell strain Chinese hamster ovary cell CHO commonly used, gene transfering efficiency is undesirable, has influenced it and has used widely.
The cell transduction peptide is that a class can be carried biomacromolecule, comprises protein, DNA, even particulate matter enters the peptide sequence of cell and subcellular structure, cry again nexin transduction domain (Proteintransduction domain, PTD).Wherein famous cell transduction peptide is the human immunodeficiency virus's (HIV) that independently finds separately of Green in 1988 and Frankel TAT albumen (Green M, Loeweastein PM.1988.Autonomous functional domain of chemicallysynthesized human immunodeficiency virus Tat transactivator protein.Cell, 55:1179-1188; Frankel AD, Pabo CO.1988.Cellular uptake of the Tat proteinfrom human immunodeficiency virus.Cell, 55:1189-1193).Further discover, TAT protein 47～57 amino acids sequences (YGRKKRRQRRR) promptly have cell transduction peptide function (Vives E, Brodin P, Lebieu is truncated HIV-1 Tat basic domainrapidly tanslocates through the plasma membrane and accumulates in the cellnucleus.J Biol Chem B.1997.A, 272:16010-16017).The cell transduction peptide is widely used in helping biomacromolecule to enter cell at present, but does not appear in the newspapers as yet with the technology that improves virus-mediated gene transfer and expression efficiency by the cell transduction peptide is illustrated on the virus nucleocapsid.
Summary of the invention
One of the object of the invention provides a kind of virus vector that improves carrier transduction and gene expression efficiency.
Two of the object of the invention provides a kind of method that improves the virus vector transduction and express goal gene efficient.
The invention provides a kind of virus vector that improves carrier transduction and gene expression efficiency, the target gene sequences that comprises viral nucleocapsid protein promoter sequence, nucleocapsid protein encoding sequence, tape starting, the encoding sequence that also comprises the cell transduction peptide in this carrier, and this cell transduction peptide is by the nucleocapsid protein promotor control transcription and translation of virus.
In the virus vector of the present invention, before the encoding sequence of this cell transduction peptide next-door neighbour nucleocapsid protein encoding sequence or afterwards.
In the virus vector of the present invention, the encoding sequence of described cell transduction peptide is positioned at after the nucleocapsid protein promoter sequence of virus, can be positioned at before the nucleocapsid protein encoding sequence.
Virus vector of the present invention can be a baculovirus, Bombyx mori nuclear polyhydrosis virus (Bombyxmori multicapsid nucleopolyhedrovirus, be called for short BmMNPV), bollworm monokaryon capsid type polyhedrosis virus (Helicoverpa armigera single nucleocapsidnucleopolyhedrovirus is called for short HaSNPV) etc.Also can comprise densonucleosis virus (Densoviruses), irido virus insect viruses and plant viruses such as (Iridoviruses), phage etc.。
In the virus vector of the present invention, described cell transduction peptide is TAT albumen the 47th～57 amino acids sequence of human immunodeficiency virus I type, VP22 (Elliott G as herpes simplex virus (HSV), O ' HareP.1997.Intercellular trafficking and protein delivery by a herpesvirus structuralprotein.Cell, 88:223-233), fruit bat transcription factor Antp (Derossi D, Joliot AH, Chassaing G, Prochiantz is third helix of the Antennapedia homeodomain translocatesthrough biological membranes.J Biol Chem A.1994.The, 269:10444-10450), preS antigen (the Peter V of H BV, Freya VH, Benedikte S, Geert L-R.2005.The arginine-richcarboxy-terminal domain of the hepatitis B virus core protein mediates attachment ofnucleocapsids to cell-surface-expressed heparan sulfate.J Gen Virol, 86:75-84) etc.
The present invention also provides the preparation method of above-mentioned virus vector, and the encoding sequence that is about to the cell transduction peptide inserts and has in the virus nucleocapsid protein carrier of target gene sequences, and the cell transduction peptide is placed under the nucleocapsid protein promotor control of virus.
For example, the proteic encoding sequence of nucleotide coding sequence and virus nucleocapsid with the cell transduction peptide is connected insertion bacterium-viral shuttle plasmid.Encoding sequence with proteic promoter sequence of virus nucleocapsid and goal gene inserts the aforementioned bacterium-viral shuttle plasmid that contains the nucleotide coding sequence and the proteic encoding sequence of virus nucleocapsid of cell transduction peptide respectively then, thus the construction of recombinant virus plasmid.
Among the present invention, the construction process of described recombinant virus is including, but not limited to the Bac-to-Bac method.Bac-to-Bac is that (a kind of commercial system of utilizing shuttle plasmid and bacterium swivel base technique construction recombinant baculovirus that USA) provides can be in bacterium be inserted foreign gene shaft-like viral genome for Carlsbad, California in Invitrogen company.
Among the present invention, the correlative coding sequence comprises because the degenerate sequence that the degeneracy of codon produces.This degenerate sequence is meant, having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so homology is low to moderate about 70% the degenerate sequence identical aminoacid sequence of also encoding out.
The present invention also provides a kind of method that improves the virus vector transduction and express goal gene efficient, promptly shows the cell transduction peptide on virus nucleocapsid.
In the aforesaid method, can be with transduction peptide and virus nucleocapsid protein fusion expression.
Among the present invention, described cell transduction peptide display location is a virus nucleocapsid, and methods of exhibiting includes but not limited to cell transduction peptide and viral natural nucleus capsid protein amalgamation and expression.Also can be with cell transduction peptide other less important nucleocapsid protein amalgamation and expressions with virus, or make the cell transduction peptide by intermolecular interaction and virus nucleocapsid in conjunction with or crosslinked etc.
Among the present invention, described virus is including, but not limited to baculovirus, its type strain autographa california nuclear polyhedrosis virus (Autographa californica multicapsidnucleopolyhedrovirus for example, be called for short AcMNPV), Bombyx mori nuclear polyhydrosis virus (Bombyx morimulticapsid nucleopolyhedrovirus, be called for short BmMNPV), bollworm monokaryon capsid type polyhedrosis virus (Helicoverpa armigera single nucleocapsidnucleopolyhedrovirus, be called for short HaSNPV) etc., also can comprise densonucleosis virus (Densoviruses), irido virus insect viruses and plant viruses such as (Iridoviruses), phage etc.
In the aforesaid method, described cell transduction peptide is including, but not limited to TAT albumen the 47th～57 amino acids sequence of human immunodeficiency virus I type, as the VP22 of herpes simplex virus (HSV), the preS antigen of fruit bat transcription factor Antp, HBV etc.
Be goal gene with mCherry (bright cherry-red fluorescin) among the present invention, the result proves that virus vector of the present invention can significantly improve transduction and the expression efficiency of goal gene in zooblast.The present invention is the external source goal gene with green fluorescent protein GFP, Luci luciferase, secretion property alkaline phosphatase SEAP etc. also, and the result shows that equally virus vector of the present invention can significantly improve transduction and the expression efficiency of goal gene in zooblast.
Raising of the present invention is transduceed in zooblast and the method for expression alien gene efficient, can be applicable to cultured cell in vitro, in tissue culture and the body, as: in cultured cell in vitro, produce protein, in vivo as the carrier of gene therapy and vaccine immunity etc.Described zooblast comprises Chinese hamster ovary cell CHO, human liver cancer cell HepG2, nasopharyngeal carcinoma cell CNE, the cultured cell in vitro system in mammalian cells such as African green monkey kidney cell VERO source, the cell of Mammals primary cultured cell and Mammals biological tissue etc.
The present invention shows the peptide chain of a kind of positive charge amino acid enrichment on virus, this peptide chain can effectively improve viral transduction efficiency.With without the virus vector of transforming relatively, the cell efficient of improved virus vector transduction is 2 times of contrast virus, in transduction back 12 hours to 96 hours, the destination gene expression level rose.The present invention is applicable to produce to have the protein of natural radioactivity, and utilizes virus vector to carry out disease gene treatment and vaccine immunity.
Description of drawings
Fig. 1. baculovirus through transforming and contrast viral genome structural representation.Acptv is for transforming back virus, and Acpv is contrast virus.
Fig. 2. the comparison diagram of baculovirus through transforming and contrast virus transduction Chinese hamster ovary cell CHO efficient.Goal gene is bright cherry-red fluorescin mCherry, and measuring method is the ratio of the back 48 hours cells of statistics generation red fluorescence under fluorescent microscope of transduction.
Fig. 3. baculovirus through transforming and contrast virus are expressed the purpose level in Chinese hamster ovary cell CHO comparison diagram.Goal gene is bright cherry-red fluorescin mCherry, and measuring method detected the average intensity of red fluorescence in back 48 hours with fluor tester for transduction.
Embodiment
Related experiment can be with reference to following steps among the present invention:
1. the structure of showing the virus vector that the cell transduction peptide is arranged: utilize Protocols in Molecular Biology, Codocyte is transduceed the dna sequence dna of peptide (as TAT, VP22, Antp, pre S antigen etc.) with viral nucleocapsid protein (as the main nucleocapsid protein VP39 of baculovirus) gene fusion, and place suitable promotor control down, obtain the expressing fusion protein sequence.Utilize swivel base (as the Bac-to-Bac system), reorganization (to recombinate as homology, Red reorganization) etc. way is incorporated into the suitable site (not influencing the site of virus replication as baculovirus polyhedrin body protein site, p10 protein loci or other) of viral genome skeleton with the expressing fusion protein sequence, obtains recombinant virus dna.
2. the acquisition of target viral: extract and transform the back recombinant virus dna, use liposome transfection, electricity transforms, CaCl
2Methods such as conversion change viral DNA in the suitable cell (as Sf9, Sf21 etc.) over to, at suitable temperature (as 27 degrees centigrade) culturing cell (being generally 2-6 days), collect cell culture fluid or cell pyrolysis liquid, promptly obtain target viral.This virus is properly preserved under appropriate condition as viral liquid of former generation.With suitable cell (as Sf9, Sf21 etc.) amplicon virus.Use the terminal point dilution method, perhaps other appropriate method is measured virus titer.For the virus of other kinds, can obtain recombinant virus with corresponding method.
3. gene transfer and expression: get target mammalian cells such as Chinese hamster ovary celI, according to tiring of viral liquid, in cell count: the ratio of viral number=1:50～200 is with virus and cytomixis, 37 degrees centigrade after incubation 4-6 hour, remove viral liquid, and add suitable fresh culture, 37 degrees centigrade are continued to cultivate 48-96 hour, detect expression conditions, or collect albumen.
Embodiment 1 obtains the purpose viral DNA
Chemosynthesis Tat (47-57aa) peptide chain corresponding DNA sequences, and merge with the dna sequence dna of coding baculovirus nucleocapsid protein VP39, insert pFastBac-1 plasmid (Invitrogen company) the EcoR I-Kpn I site of Bac-to-Bac system, obtain pFB-tv.Pcr amplification obtains vp39 promoter DNA sequence and is inserted into pFB-tv plasmid EcoR I site, obtains pFB-ptv.Insert the CMV-mCherryDNA sequence in Sal I site, obtain plasmid pFB-ptvc.Utilize similar method to obtain control plasmid pFB-pvc, in this plasmid, natural vp39 replaces having merged the tat-vp39 of Tat.Utilize the Bac-to-Bac system to obtain recombinant virus dna.
Embodiment 2 obtains purpose virus
With the purpose recombinant virus dna transfection Sf9 cell that obtains, obtain viral and amplicon virus of former generation, mensuration is tired.Target viral is named as Acptv and Acpv (Fig. 1).
Embodiment 3 expressing proteins
With 37 degrees centigrade of overnight incubation of Chinese hamster ovary celI, remove nutrient solution, the PBS washed twice, according to cell count: viral number=1:50 adds viral liquid, 37 degrees centigrade of incubations 6 hours remove viral liquid, and with twice of PBS washed cell, 37 degrees centigrade leave standstill cultivation 48h, detect the cells ratio (Fig. 2) and the cell average fluorescent strength (Fig. 3) that produce fluorescence.
It is the external source goal gene that embodiment 4 adopts green fluorescent protein GFP
According to the method for embodiment 1-3, with the dna sequence dna replacement mCherryDNA sequence of green fluorescent protein GFP, construction of recombinant virus; Then, transfection Sf21 cell amplification virus; Subsequently, in Chinese hamster ovary celI according to cell count: the ratio of viral number=1:25 adds viral liquid; Detect at last, the cells ratio and the cell average fluorescent strength that produce green fluorescence all are more than 2 times of contrast.
Claims (9)
1, a kind of virus vector that improves carrier transduction and gene expression efficiency, the target gene sequences that comprises viral nucleocapsid protein promoter sequence, nucleocapsid protein encoding sequence, tape starting, it is characterized in that, the encoding sequence that also comprises the cell transduction peptide in this carrier, and before the encoding sequence of this cell transduction peptide next-door neighbour nucleocapsid protein encoding sequence or afterwards.
2, virus vector as claimed in claim 1 is characterized in that, the encoding sequence of described cell transduction peptide is positioned at after the viral nucleocapsid protein promoter sequence and is positioned at before the nucleocapsid protein encoding sequence.
As claim 1 or 2 described virus vector, it is characterized in that 3, described virus is baculovirus, Bombyx mori nuclear polyhydrosis virus or bollworm monokaryon capsid type polyhedrosis virus.
4, as claim 1 or 2 described virus vector, it is characterized in that described cell transduction peptide is TAT albumen the 47th～57 amino acids sequence of human immunodeficiency virus I type, VP22, the fruit bat transcription factor Antp of herpes simplex virus or the preS antigen of HBV.
5, as the preparation method of virus vector as described in claim 1 or 2, it is characterized in that, the encoding sequence insertion of cell transduction peptide is had in the virus nucleocapsid protein carrier of target gene sequences, and the cell transduction peptide is placed under the nucleocapsid protein promotor control of virus.
6, a kind of method that improves virus vector transduction and expressing gene efficient is characterized in that, shows the cell transduction peptide on virus nucleocapsid.
7, method as claimed in claim 6 is characterized in that, will transduce peptide and virus nucleocapsid protein fusion expression.
8, method as claimed in claim 6 is characterized in that, described virus is baculovirus, Bombyx mori nuclear polyhydrosis virus, bollworm monokaryon capsid type polyhedrosis virus, densonucleosis virus, irido virus or phage.
9, method as claimed in claim 6 is characterized in that, described cell transduction peptide is TAT albumen the 47th～57 amino acids sequence of human immunodeficiency virus I type, VP22, the fruit bat transcription factor Antp of herpes simplex virus or the preS antigen of HBV.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059153A (en) * | 2013-09-13 | 2014-09-24 | 西安交通大学医学院第一附属医院 | Transmembrane antibody of targeting virus core protein and preparation method thereof |
| CN105200016A (en) * | 2015-09-23 | 2015-12-30 | 中国检验检疫科学研究院 | DNA virus reference material and preparation method and application thereof |
| CN108588122A (en) * | 2018-04-18 | 2018-09-28 | 西南大学 | The polygenes element and expression vector and application that two gene equivalent are expressed are realized based on 2A cleavage of peptide |
| CN111387213A (en) * | 2020-03-24 | 2020-07-10 | 湖北大学 | Preparation method of virus-like particles for preventing and treating cotton bollworm, method for resisting cotton bollworm and application of virus-like particles |
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2008
- 2008-12-18 CN CNA2008102072066A patent/CN101429524A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059153A (en) * | 2013-09-13 | 2014-09-24 | 西安交通大学医学院第一附属医院 | Transmembrane antibody of targeting virus core protein and preparation method thereof |
| CN105200016A (en) * | 2015-09-23 | 2015-12-30 | 中国检验检疫科学研究院 | DNA virus reference material and preparation method and application thereof |
| CN108588122A (en) * | 2018-04-18 | 2018-09-28 | 西南大学 | The polygenes element and expression vector and application that two gene equivalent are expressed are realized based on 2A cleavage of peptide |
| CN111387213A (en) * | 2020-03-24 | 2020-07-10 | 湖北大学 | Preparation method of virus-like particles for preventing and treating cotton bollworm, method for resisting cotton bollworm and application of virus-like particles |
| CN111387213B (en) * | 2020-03-24 | 2021-06-29 | 湖北大学 | Preparation method of virus-like particles for preventing and treating cotton bollworm, method for resisting cotton bollworm and application of virus-like particles |
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Open date: 20090513 |