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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (14): 110-113.doi: 10.11924/j.issn.1000-6850.2011-2249

所属专题： 生物技术

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

A型口蹄疫病毒VP1基因的克隆及原核表达

刘明 刘航 柳纪省   

  • 收稿日期:2011-08-02 修回日期:2011-09-14 出版日期:2012-05-15 发布日期:2012-05-15

Cloning and Prokaryotic expression of VP1 Gene of Foot-and-Mouth Disease Virus (FMDV) Type A

  • Received:2011-08-02 Revised:2011-09-14 Online:2012-05-15 Published:2012-05-15

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摘要：

文章旨在利用原核表达系统表达并纯化出A型口蹄疫病毒VP1蛋白。首先从实验室冻存的含A型口蹄疫病毒的细胞中提取FMDV总RNA，通过RT-PCR获得cDNA。并根据FMDV全基因组序列设计了一对针对VP1基因的引物，通过PCR扩增得到目的基因VP1并亚克隆入pMD 18-T载体。将鉴定出的阳性质粒和表达载体PET32a用BamHⅠ和Hind Ⅲ双酶切回收后连接获得阳性重组质粒PET32-VP1。用IPTG诱导重组质粒表达目的蛋白VP1并用SDS-PAGE进行检测。表达产物用镍亲和树脂进行了纯化。结果证明A型口蹄疫病毒VP1蛋白在大肠杆菌中获得了高效表达且表达产物得到了纯化，为实验室进一步的研究提供了重要的材料。

关键词: 大白菜, 大白菜, 组织培养, 遗传转化, 正交试验

Abstract:

Extract the RNA of FMDV tape A from frozen cells in the laboratory,then acquire the cDNA by RT-PCR. A special primer pair was designed which containing BamHⅠand HindⅢ according to complete genome of FMDV,the target gene VP1 was amplified by PCR and subcloned into pMD 18-T vector.Then, digest the VP1 from pMD18-T vector and expression vector PET32a with BamHⅠand HindⅢ, ligated gene VP1 into PET32a and named recombinant plasmid PET32-VP1 after identification. The interest gene was induced to express in E.coli with IPTG and identified with SDS-PAGE.The target protein was purified with Ni-NTA Purification System. Result showed that the target protein was expressed in E.coli and also purified.This provide laboratory important material for further study.