文档介绍：
EV71 衣壳蛋白基因克隆与表达#
梁婕1,罗海华1,刘爱华2,姜勇1**
(1. 广州南方医科大学病理生理学教研室暨广东省蛋白质组学重点实验室,广州 510515;
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2. 广州南方医科大学南方医院呼吸科,广州 510515)
摘要:目的:构建 EV71 衣壳蛋白基因的原核表达载体,并在原核细胞中进行表达与纯化。
方法:根据已知 EV71 全长核酸序列设计引物,用 PCR 方法扩增 VP1、VP2、VP3 和 VP4
四个基因片段,对目的基因进行酶切并克隆至 pET-14b 原核表达载体中,转化大肠杆菌
BL21(DE3)感受态细胞,IPTG 诱导融合蛋白 VP1~VP4 表达;所获得的不可溶性蛋白经尿素
裂解超声,亲和层析纯化,用 SDS-PAGE 检测表达产物。结果:测序证实原核重组质粒构
建成功;表达纯化的蛋白主要以包涵体存在,经变性复性可得到高纯度的 VP4 蛋白,
VP1~VP3 蛋白表观质量与理论预期有一定差异且纯度较低。结论:成功构建了手足口病主
要致病原肠道病毒 71 型(EV71)的四个衣壳蛋白 VP1、VP2、VP3 和 VP4 的原核表达载体,
纯化得到高纯度的 VP4 蛋白,为进一步研究 EV71 衣壳蛋白致病机理奠定了基础。
关键词:EV71;基因克隆;蛋白表达
中图分类号:
Gene cloning and expression of capsid proteins of EV71
LIANG Jie1, LUO Haihua1, LIU Aihua2, JIANGYong1
(1. Department of Pathophysiology, Key Laboratory of Proteomics of Gongdong Province,
Southern Medical University, Guangzhou 510515;
2. Southern Medical University, Nanfang Hospital, Guangzhou 510515)
Abstract: Objective To express and purify the capsid proteins, . VP1, VP2, VP3 and VP4 of
EV71 in E. coli, which mainly caused hand, foot and mouth disease. Methods According to the
full-length gene sequence of EV71 reported in GenBank, the primers were designed by using
DNA Star software, based on which VP1-4 genes were amplified by PCR and inserted into
prokaryotic expression vector pET-14b respectively. The constructed binant plasmid
pET-14b-VPs were transformed to E.