零背景转座技术高效构建重组家蚕杆状病毒表达系统  

Construction of recombinant Bombyx mori Nucleopolyhedrovirus by zero background Tn7 transposition

作　　者：姚伦广[1] 张红玲[1,2] 冯娟[1,3] 张二辉[1] 文祯中[1,2] 

机构地区：[1]中英南阳洛桑昆虫生物学联合实验室,河南省伏牛山昆虫生物学重点实验室,南阳师范学院生物科学与技术学院,南阳473061 [2]河南师范大学生命科学学院,新乡453007 [3]河南农业大学生命科学学院,郑州450002

出　　处：《微生物学报》2009年第6期831-837,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30700750);南阳市重大科技攻关项目(2007sy001);河南省教育厅科技攻关项目(2008A180020)~~

摘　　要：【目的】获得零转座背景的基于家蚕核型多角体病毒(Bombyx mori Nucleopolyhedrovirus,BmNPV)Bac-to-Bac系统,为高效经济构建重组BmNPV在家蚕体内表达目标蛋白提供新系统。【方法】利用R6Kγ作为复制子构建新的条件复制型杆状病毒转移载体pRADM,同时封闭BmNPV-Bacmid(Bm Bacmid)宿主菌(Escherichia coli BmDH10 Bac)的Tn7转座受体位点attTn7,获得新的封闭型宿主菌E.coli BmDH10 Bac△Tn7。【结果】由于pRADM无法在宿主菌E.coliBmDH10Bac中复制,封闭了attTn7位点的宿主菌也不能再和BmBacmid竞争与转移载体的重组,显著提高了转座效率。封闭宿主菌的attTn7位点,能使转座效率提高近4倍,使用条件复制型转座载体pRADM时,转座效率提高近10倍。而用pRADM转座E.coli BmDH10 Bac△Tn7时,转座阳性率为100%。避免了获得重组病毒DNA的鉴定程序,缩短了获得重组蛋白所需时间。用携带红色荧光蛋白基因DsRed的重组质粒pRADM-Red转座E.coli BmDH10 Bac△Tn7,获得重组BmBacmid转染BmN细胞,红色荧光蛋白在细胞中得到高效表达。【结论】结果表明pRADM和E.coli BmDH10 Bac△Tn7是一种零背景高效构建重组BmNPV的新系统。[ Objective] In order to construct a recombinant Bombyx mori Nucleopolyhedrovirus by Tn7-mediated transposition in Escherichia coli efficiently, a new zero background transposition system was developed. [ Method] The new system consisted of a conditional replication donor vector pRADM and an attTn7 site blocked E. coli containing BmNPV-Baemid. The donor transposon vector pRADM with the replication origin derived from R6Kγ required the factor π encoded by the pir gene to propagate in host ceils. Another conditional replication plasmid pBlockA was constructed to block the attTn7 site in host E. coli genome. [ Results] Compared with the original vector with ColE1 origin, the transposition efficiency increased from 5.7 % to 66% when using conditional replication vector pRADM transposition into original BmDH10Bac. The attTn7 site blocked strain BmDH10Bac△Tn7 resulted in a significant increase from 5.7% to 23% in the efficacy of generating recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10Bac△Tn7 with pRADM resulted in 100% white colonies. [ Conclusion] This highly efficient and zero background transposition system provides a simple and rapid way of construction of recombinant BmNPV to express target genes or produce gene-delivery virus particles in silkworm.

关 键 词：条件复制子 零背景转座 重组家蚕核型多角体病毒 

分 类 号：Q786[生物学—分子生物学]