摘要
通过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HindⅢ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。
The article aims to express and purified VP1 protein of FMDV tape A using prokaryotic expression system.First 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.
出处
《中国农学通报》
CSCD
2012年第14期110-113,共4页
Chinese Agricultural Science Bulletin
基金
中央级公益性科研院所基本科研业务费课题(BRF070307)
关键词
口蹄疫病毒
VP1基因
原核表达
foot-and-mouth disease virus
VP1gene
prokaryotic expression
