创建可视化的家蚕杆状病毒表达系统表达家蚕二分浓核病毒非结构蛋白NS1

重组杆状病毒感染昆虫细胞是表达外源蛋白常用的一种方法。为有效鉴定转染的细胞中是否产生了重组病毒粒子,对质粒pFastBacI进行改造,构建了极早期基因ie1启动子控制的绿色荧光蛋白egfp基因表达盒,以及多角体基因启动子控制的外源DNA的一个通用型双表达载体;通过酶切、连接的方式,将家蚕二分浓核病毒ns1基因连接到多角体启动子下游;在转座酶的介导下,该供体质粒上部分序列可转座到穿梭载体Bm-Bacmid上,进而构建可同时表达egfp和ns1基因的重组杆粒。将构建的该重组杆粒DNA转染BmN细胞,通过观察可视化的绿色荧光信号,可迅速判定转染后的细胞中重组病毒粒子产生的情况,收集转染后的细胞培养上清,将其感染BmN细胞,对感染4 d后的细胞总蛋白进行Western blotting分析,结果表明能杂交到一条36 kDa大小的特异蛋白,表明NS1蛋白成功获得了表达,进而为深入研究ns1基因的功能奠定了基础。

Expression of ns1 gene from Bombyx mori bidensovirus by a novel Baculovirus expression system

Baculovirus gene expression is the most popular method to make target protein in cultured insect cells. To fast determine the generation of recombinant virus in cultured cells, donor plasmid of pFastBacI was modified by introducing egfp cassette. In the modified vector, egfp cassette was under the control of ie1 promoter, and target gene cassette was under the control of polyhedron promoter. To evaluate the convenience of the genetically modified donor plasmid used in eukaryotic expression, ns1 gene from Bombyx mori bidensovirus was ligated into the donor plasmid to generate recombinant plasmid pFastBacI-Pie1-egfp-sv40-Ppolh-ns1-sv40. Then the plasmid was transformed into DH10B competent cells containing Bm-Bacmid vector to produce the final recombinant Bm-Bacmid with the help of transposase. The resulting recombinant Bm-Bacmid was transfected into BmN cells to generate recombinant virus, which was easily and rapidly judged by green fluorescent signal observed in BmN cells. After infection for 96 h, the BmN cells were harvested and the total protein extracted from the infected BmN cells was subjected to Western blotting analysis. The result shows that a specific protein band about 36 kDa was detected, indicating that NS1 protein was successfully expressed in the BmN cells. In conclusion, the expression of NS1 protein with the modified expression system is useful for further research on the function of NS1 protein.