[1]肖圣燕,李镇刚,冉瑞法,等.家蚕微孢子虫SWP25基因BmN细胞表达质粒的构建及表达[J].江苏农业学报,2016,(05):1023-1028.[doi:10.3969/j.issn.1000-4440.2016.05.011]
　XIAO Sheng-yan,LI Zhen-gang,RAN Rui-fa,et al.Construction of the eukaryotic expression plasmid containing 〖WTHX〗SWP25〖WTHZ〗 gene of Nosema bombycis and its expression in silkworm BmN cell[J].,2016,(05):1023-1028.[doi:10.3969/j.issn.1000-4440.2016.05.011]
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家蚕微孢子虫SWP25基因BmN细胞表达质粒的构建及表达()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2016年05期
页码:
1023-1028
栏目:
遗传育种·生理生化
出版日期:
2016-11-22

文章信息/Info

Title:
Construction of the eukaryotic expression plasmid containing 〖WTHX〗SWP25〖WTHZ〗 gene of Nosema bombycis and its expression in silkworm BmN cell
作者:
肖圣燕1; 李镇刚1; 冉瑞法1; 黄平1; 朱峰1; 沈中元2; 3
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
Author(s):
XIAO Sheng-yan1; LI Zhen-gang1; RAN Rui-fa1; HUANG Ping1; ZHU Feng1; SHEN Zhong-yuan2; 3
1. Institute of Sericulture and Apiculture, Yunnan Academy of Agricultural Sciences, Mengzi 661101, China;2. Jiangsu University of Science and Technology，Zhenjiang 212003, China;3. The Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China
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
Keywords:
Nosema bombycis;  spore wall protein;  Bac-to-Bac baculovirus expression system;  recombinant plasmid;  transfection;  expression of fusion protein
分类号:
S884.2
DOI:
10.3969/j.issn.1000-4440.2016.05.011
文献标志码:
A
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pFastBac1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vBmEGFP-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。
Abstract:
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 vBmEGFP-SWP25 bacmid was identified by PCR technique. A uorescence signal was observed in the transfected BmN cells. Western-blot analysis revealed a protein band of approximately 55 000 in vBm EGFP-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 successfully expressed in BmN cells.

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备注/Memo

备注/Memo:
收稿日期：2016-02-15 基金项目：云南省农业科学院蚕桑蜜蜂研究所青年创新基金项目（QC2015001） 作者简介：肖圣燕（1989-），女，山东泰安人，硕士，研究实习员，主要从事桑树资源与育种研究。（Tel）15187388921；（E-mail）shengyanxiao1989@163.com 通讯作者：沈中元，（Tel）0511-85616665；（E-mail）szysri@163.com
更新日期/Last Update: 2016-11-22