利用细菌转座子构建适于家蚕的Bac-to-Bac快速基因表达系统

为了较好地解决家蚕杆状病毒基因表达系统(BmNPV expression system)在重组病毒构建和纯化过程中存在的重组率低、空斑分析技术繁琐、花费时间长等缺点,借鉴国外AcNPV Bac-to-Bac快速基因表达系统工作原理,对家蚕BmNPV基因组进行了改造。通过同源重组的方法,将含有单拷贝数细菌F复制子、插入有细菌转座子整合靶位点、编码LacZα肽的部分DNA片段和抗性选择标记基因的基因片段重组入家蚕BmNPV基因组,替换多角体蛋白基因,获得了家蚕BmNPV病毒穿梭载体BmBacm id;并利用供体质粒上的表达盒和细菌转座子以及细菌转座子的基因定位转移作用,在细菌体内实现外源目的基因向家蚕BmNPV基因组上的转移整合,快速完成重组BmN-PV病毒的构建。

Construction of A Rapid BmNPV Bac-to-Bac System for Application in Silkworm, Bombyx mori L. by Using Bacterial Transposon

In order to overcome some disadvantages of BmNPV expression system,such as low recombination frequency,laborious plaque assays and long-time taking,a novel and rapid expression system based on the principle of AcNPV Bac-to-Bac system was constructed in this study.This system can generate recombinant baculovirus genome in bacterium through gene transposition by transposon,and can be applied in silkworm.In this construction,a large DNA fragment containing the low-copy-number mini-F replicon,a kanamycin resistance marker,and a segment of DNA encoding the lacZα peptide was inserted into the locus of polyhedrin gene of BmNPV genome.Thus obtained recombinant BmNPV genome was named as BmBacmid.To produce the recombinant BmNPV,the foreign gene was first subcloned into the donor vector which equipped with bacterial transposon,and subsequently transposed to BmBacmid.By color selection,the recombinant BmBacmid,i.e.,the recombinant baculovirus genome was rapidly isolated,and thereafter the recombinant baculovirus was easily generated through transfection into cultured cells.