复制缺陷型BmNPV载体的构建及初步应用
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摘要
家蚕核型多角体病毒(BmNPV)表达载体系统可以利用线性化技术或通过Bac-to-Bac系统构建重组病毒。为了解决采用这些方法构建重组病毒需要多步克隆和筛选,无法满足实验室水平快速和高通量表达目的蛋白质需求的问题,建立了一种快速便捷的重组病毒构建方法。首先以BmNPV基因组为基础构建家蚕杆状病毒穿梭载体BmBacmid,进而利用Red重组技术敲除病毒复制必需基因orf1629的部分序列,构建了一种复制缺陷型BmNPV穿梭载体RD-BmBacmid。利用RD-BmBac-mid单独转染或者与pBacPAK8-AcGFP共转染BmN细胞,证实了该载体的复制缺陷性,即需经同源重组修复orf1629基因后方可产生重组病毒。利用复制缺陷型BmNPV载体正筛选可以快速构建重组病毒表达目的蛋白质。
Bombyx mori nucleopolyhedrovirus(BmNPV) expression vector system can be used to construct recombinant virus using linearization technology or Bac-to-Bac system,both of which require multiple sub-cloning and screening,being not able to satisfy the demand of quick and high throughput expression of target protein in laboratories.Herein,we developed a simple and rapid approach to construct recombinant virus.Firstly,a Bombyx mori baculovirus shuttle vector BmBacmid was constructed based on BmNPV genome.Then,part of the replication essential gene orf1629 was knocked out using Red recombination technology to construct the replication-defective BmNPV shuttle vector RD-BmBacmid.Single transfection with RD-BmBacmid or co-transfection with pBacPAK8-AcGFP into BmN cells verified replication deficiency of this vector,meaning that recovery of virus replication required orf1629 gene repair through homologous recombination.Utilization of positive screening to this replication-defective BmNPV vector enables quick construction of recombinant virus for expressing target protein.
Bombyx mori nucleopolyhedrovirus(BmNPV) expression vector system can be used to construct recombinant virus using linearization technology or Bac-to-Bac system,both of which require multiple sub-cloning and screening,being not able to satisfy the demand of quick and high throughput expression of target protein in laboratories.Herein,we developed a simple and rapid approach to construct recombinant virus.Firstly,a Bombyx mori baculovirus shuttle vector BmBacmid was constructed based on BmNPV genome.Then,part of the replication essential gene orf1629 was knocked out using Red recombination technology to construct the replication-defective BmNPV shuttle vector RD-BmBacmid.Single transfection with RD-BmBacmid or co-transfection with pBacPAK8-AcGFP into BmN cells verified replication deficiency of this vector,meaning that recovery of virus replication required orf1629 gene repair through homologous recombination.Utilization of positive screening to this replication-defective BmNPV vector enables quick construction of recombinant virus for expressing target protein.
引文
[1]Galleno M,Sick A I.Baculovirus expression vector system[M]∥Fernandez J M,Hoeffler J P.Gene expression systems:using naturefor the art of expression.San Diego:Academic Press,1999:331-361
[2]Smith G E,Summers M D,Fraser M J.Production of human betainterferon in insect cells infected with a baculovirus expression vec-tor[J].Mol Cell Biol,1983,3(12):2156-2165
[3]Hitchman R B,Possee R D,King L A.Baculovirus expression sys-tems for recombinant protein production in insect cells[J].RecentPat Biotechnol,2009,3(1):46-54
[4]韦永龙,李轶女,张志芳,等.杆状病毒表达系统及其应用进展[J].生物技术通报,2010,13(10):1-7
[5]Wu X F,Cao C P,Xu Y X,et al.Construction of a host range-ex-panded hybrid baculovirus of BmNPV and AcNPV,and knockout ofcysteinase gene for more efficient expression[J].Sci China C,LifeSci,2004,47(5):406-415
[6]Maeda S,Kawai T,Obinata M,et al.Production of humanα-inter-feron in silkworm using a baculovirus vector[J].Nature,1985,315(6020):592-594
[7]Wu X F,Yang G Z,Hu J X.A recombinant rescue linearizableBmNPV baculovirus BmBacPAK:China,98110963.2[P].1998-11-09
[8]Motohashi T,Shimojima T,Fukagawa T,et al.Efficient large-scaleprotein production of larvae and pupae of silkworm by Bombyx morinuclear polyhedrosis virus bacmid system[J].Biochem BiophysRes Commun,2005,326(3):564-569
[9]Je Y H,Chang J H,Kim M H,et al.The use of defective Bombyxmori nucleopolyhedrovirus genomes maintained in Escherichia colifor the rapid generation of occlusion-positive and occlusion-negativeexpression vectors[J].Biotechnol Lett,2001,23(21):1809-1817
[10]Lee K S,Kim B Y,Je Y H,et al.A new technique for producingrecombinant baculovirus directly in silkworm larvae[J].BiotechnolLett,2007,29(1):175-180
[11]Li X H,Wang M X,Zhang P,et al.Heterologous expression char-acteristics of Trichoderma viride endoglucanase V in the silkworm,Bombyx mori L.[J].Appl Biochem Biotechnol,2011,165(2):728-736
[12]Zhou L,Wu X F,Lan L P,et al.Expression of Trichoderma reeseiendo-beta-glucanaseⅡin silkworm,Bombyx mori L.by using Bm-NPV/Bac-to-Bac expression system and its bioactivity assay[J].Biotechnol Lett,2010,32(1):67-72
[13]Yao L G,Sun J C,Xu H,et al.A novel economic method for highthroughput production of recombinant baculovirus by infecting in-sect cells with Bacmid-containing diminopimelate-auxotrophicEscherichia coli[J].J Biotechnol,2010,145(1):23-29
[14]Ono C,Kamagata T,Taka H,et al.Phenotypic grouping of 141 Bm-NPVs lacking viral gene sequences[J].Virus Res,2012,165(2):197-206
[15]Yao L G,Wang S S,Su S,et al.Construction of a baculovirus-silk-worm multigene expression system and its application on producingvirus-like particles[J/OL].PLoS One,2012,7(3):e32510[2013-01-12].http:∥www.plosone.org/article/info%3Aoi%2F10.1371%2Fjournal.pone.0032510
[16]Monaco A P,Larin Z.YACs,BACs,PACs and MACs:artificialchromosomes as research tools[J].Trends Biotechnol,1994,12(7):280-286
[17]Pijlman G P,Van Schijndel J E,Vlak J M.Spontaneous excision ofBAC vector sequences from bacmid-derived baculovirus expressionvectors upon passage in insect cells[J].J Gen Virol,2003,84(Pt10):2669-2678
[18]Hitchman R B,Siaterli E A,Nixon C P,et al.Quantitative real-time PCR for rapid and accurate titration of recombinant baculovir-us particles[J].Biotechnol Bioeng,2007,96(4):810-814
[2]Smith G E,Summers M D,Fraser M J.Production of human betainterferon in insect cells infected with a baculovirus expression vec-tor[J].Mol Cell Biol,1983,3(12):2156-2165
[3]Hitchman R B,Possee R D,King L A.Baculovirus expression sys-tems for recombinant protein production in insect cells[J].RecentPat Biotechnol,2009,3(1):46-54
[4]韦永龙,李轶女,张志芳,等.杆状病毒表达系统及其应用进展[J].生物技术通报,2010,13(10):1-7
[5]Wu X F,Cao C P,Xu Y X,et al.Construction of a host range-ex-panded hybrid baculovirus of BmNPV and AcNPV,and knockout ofcysteinase gene for more efficient expression[J].Sci China C,LifeSci,2004,47(5):406-415
[6]Maeda S,Kawai T,Obinata M,et al.Production of humanα-inter-feron in silkworm using a baculovirus vector[J].Nature,1985,315(6020):592-594
[7]Wu X F,Yang G Z,Hu J X.A recombinant rescue linearizableBmNPV baculovirus BmBacPAK:China,98110963.2[P].1998-11-09
[8]Motohashi T,Shimojima T,Fukagawa T,et al.Efficient large-scaleprotein production of larvae and pupae of silkworm by Bombyx morinuclear polyhedrosis virus bacmid system[J].Biochem BiophysRes Commun,2005,326(3):564-569
[9]Je Y H,Chang J H,Kim M H,et al.The use of defective Bombyxmori nucleopolyhedrovirus genomes maintained in Escherichia colifor the rapid generation of occlusion-positive and occlusion-negativeexpression vectors[J].Biotechnol Lett,2001,23(21):1809-1817
[10]Lee K S,Kim B Y,Je Y H,et al.A new technique for producingrecombinant baculovirus directly in silkworm larvae[J].BiotechnolLett,2007,29(1):175-180
[11]Li X H,Wang M X,Zhang P,et al.Heterologous expression char-acteristics of Trichoderma viride endoglucanase V in the silkworm,Bombyx mori L.[J].Appl Biochem Biotechnol,2011,165(2):728-736
[12]Zhou L,Wu X F,Lan L P,et al.Expression of Trichoderma reeseiendo-beta-glucanaseⅡin silkworm,Bombyx mori L.by using Bm-NPV/Bac-to-Bac expression system and its bioactivity assay[J].Biotechnol Lett,2010,32(1):67-72
[13]Yao L G,Sun J C,Xu H,et al.A novel economic method for highthroughput production of recombinant baculovirus by infecting in-sect cells with Bacmid-containing diminopimelate-auxotrophicEscherichia coli[J].J Biotechnol,2010,145(1):23-29
[14]Ono C,Kamagata T,Taka H,et al.Phenotypic grouping of 141 Bm-NPVs lacking viral gene sequences[J].Virus Res,2012,165(2):197-206
[15]Yao L G,Wang S S,Su S,et al.Construction of a baculovirus-silk-worm multigene expression system and its application on producingvirus-like particles[J/OL].PLoS One,2012,7(3):e32510[2013-01-12].http:∥www.plosone.org/article/info%3Aoi%2F10.1371%2Fjournal.pone.0032510
[16]Monaco A P,Larin Z.YACs,BACs,PACs and MACs:artificialchromosomes as research tools[J].Trends Biotechnol,1994,12(7):280-286
[17]Pijlman G P,Van Schijndel J E,Vlak J M.Spontaneous excision ofBAC vector sequences from bacmid-derived baculovirus expressionvectors upon passage in insect cells[J].J Gen Virol,2003,84(Pt10):2669-2678
[18]Hitchman R B,Siaterli E A,Nixon C P,et al.Quantitative real-time PCR for rapid and accurate titration of recombinant baculovir-us particles[J].Biotechnol Bioeng,2007,96(4):810-814