痘苗病毒/T7 RNA聚合酶辅助的原核基因在真核细胞中的表达(英文)  

Expression of Prokaryotic Gene in Mammalian Cells Based on Recombinant Vaccinia Virus Encoding T7 RNA Polymerase

作　　者：潘小霞[1] 张顺[1] 文喻玲[1] 袁静[1] 陈元鼎[1] 

机构地区：[1]中国医学科学院&北京协和医学院医学生物学研究所昆明650118

出　　处：《中国生物工程杂志》2011年第5期48-54,共7页China Biotechnology

基　　金：Supported by a grant from Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College in 2009 (IMB2009YB02)~~

摘　　要：痘苗病毒/T7RNA聚合酶这一瞬时表达系统由于具有很多优于其他表达系统的特点而被广泛地应用于表达外源蛋白。TB-Chen株轮状病毒的VP6 DNA编码片段插入到原核质粒pETL的噬菌体T7启动子和终止子之间,获得重组表达质粒pET-VP6。构建好的重组表达质粒pET-VP6通过脂质体转染到真核细胞MA104中,用携带噬菌体T7RNA聚合酶基因的重组痘苗病毒vTF7-3再感染,可在细胞检测到目的蛋白VP6的表达。研究结果还显示,痘苗病毒vTF7-3感染后,细胞病变较快,严重影响了蛋白的表达量。结果提示,如果能降低痘苗病毒vTF7-3的致细胞病变作用而不影响其在细胞中合成T7 RNA聚合酶的能力,蛋白可能会得到高效表达。为进一步研究VP6基因的特性及更好地应用痘苗病毒/T7 RNA聚合酶这一瞬时表达系统提供理论基础。The vaccinia virus/T7 RNA polymerase transient expression system relying on the bacteriophage T7 RNA polymerase with several key advantages was widely used for protein expression.VP6-encoding cDNA of rotavirus strain TB-Chen was cloned and constructed by inserting into prokaryotic expression plasmid pETL between bacteriophage T7 promoter and terminator regions.When eukaryotic cells were transfected with the recombinant plasmid containing the VP6 gene and infected with recombinant vaccinia virus vTF7-3 which provides bacteriophage T7 RNA polymerase,the VP6 gene was expressed.The findings showed that cytopathic effects by vaccinia virus infection were fast and severe leading to low level of protein expression.If cytopathic effects of the vaccinia virus can be reduced,higher level of the protein might be obtained.The findings provided a helpful means to further study the VP6 function and utilize the vaccinia virus/T7 RNA polymerase transient expression system.

关 键 词：轮状病毒VP6 原核质粒表达 真核细胞 重组痘苗病毒vTF7-3 T7 RNA聚合酶 

分 类 号：Q786[生物学—分子生物学]