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

摘要:

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（BmBacmid）宿主菌（Escherichia coli BmDH10Bac）的Tn7转座受体位点attTn7，获得新的封闭型宿主菌E.coli BmDH10Bac△Tn7。【结果】由于pRADM无法在宿主菌E.coli BmDH10Bac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 BmDH10Bac△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 BmDH10Bac△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 BmDH10Bac△Tn7是一种零背景高效构建重组BmNPV的新系统。

Abstract:

Abstract: [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-Bacmid. 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 cells. 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.