家蚕微孢子虫SWP25基因BmN细胞表达质粒的构建及表达  

Construction of the eukaryotic expression plasmid containing SWP25 gene of Nosema bombycis and its expression in silkworm Bm N cell

作　　者：肖圣燕 李镇刚[1] 冉瑞法[1] 黄平[1] 朱峰[1] 沈中元[2,3] XIAO Sheng-yan LI Zhen-gang RAN Rui-fa HUANG Ping ZHU Feng SHEN Zhong-yuan(Institute of Sericulture and Apiculture, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China Jiangsu University of Science and Tech- nology , Zhenjiang 212003, China The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China)

机构地区：[1]云南省农业科学院蚕桑蜜蜂研究所,云南蒙自661101 [2]江苏科技大学,江苏镇江212003 [3]中国农业科学院蚕业研究所,江苏镇江212018

出　　处：《江苏农业学报》2016年第5期1023-1028,共6页Jiangsu Journal of Agricultural Sciences

基　　金：云南省农业科学院蚕桑蜜蜂研究所青年创新基金项目(QC2015001)

摘　　要：本研究旨在构建含家蚕微孢子虫孢壁蛋白基因SWP25的家蚕卵巢细胞(BmN)表达质粒,检测该基因在宿主细胞中的表达,为在细胞水平筛选与家蚕微孢子虫孢壁蛋白SWP25相互作用的宿主蛋白奠定基础。利用家蚕核型多角体病毒Bac-to-Bac表达系统,将家蚕微孢子虫孢壁蛋白基因SWP25和报告基因EGFP克隆到杆状病毒转移载体pFast Bac1中,转化BmDH10Bac感受态细胞并筛选阳性重组质粒用于转染BmN细胞。经PCR技术鉴定,成功获得重组杆状病毒质粒v BmEGFP-SWP25。在转染成功的细胞中可观测到绿色荧光信号。使用Western blot方法在感染重组病毒的家蚕BmN细胞中检测到大小约55 000的重组蛋白条带,与理论值一致,表明家蚕微孢子虫孢壁蛋白基因SWP25在BmN中成功表达。This study aims to construct the eukaryotic expression plasmid containing SWP25 gene of Nosema bombycis and detect its expression in host cells （ BmN ） for further researches on screening host protein interacting with SWP25. The SWP25 and EGFP genes were inserted into the baculovirus transfer vector pFastBac1 to convert BmDH10Bac cells and to screen positive recombinant plasmid for transfecting BmN cells. Successful achievement of the recombinant vBmEGFP-SWP25 bacmid was identified by PCR technique. A fluorescence signal was observed in the transfected BmN cells. Western-blot analysis revealed a protein band of approximately 55 000 in vBmEGFP-SWP25 transfected BmN cells, which was consistent with the deduced molecular weight of the egfp-SWP25 fusion protein. It was concluded that SWP25 had been suc-cessfully expressed in BmN cells.

关 键 词：家蚕微孢子虫 孢壁蛋白SWP25基因 Bac-to-Bac表达系统 重组质粒 转染 融合蛋白表达 

分 类 号：S884.2[农业科学—特种经济动物饲养]