痘苗病毒/T7RNA聚合酶辅助的原核基因在真核细胞中的表达

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...

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

The vaccinia virus/T7 RNA polymerase transient expression system relying on the bacteriophageT7 RNA polymerase with several key advantages was widely used for protein expression. VP6-encoding cDNA ofrotavirus strain TB-Chen was cloned and constructed by inserting into prokaryotic expression plasmid pETL betweenbacteriophage T7 promoter and terminator regions. When eukaryotic cells were transfeeted with the recombinantplasmid containing the VP6 gene and infected with recombinant vaccinia virus vTF7-3 which provides bacteriophageT7 RNA polymerase, the VP6 gene was expressed. The findings showed that cytopathic effects by vaccinia virusinfection were fast and severe leading to low level of protein expression. If cytopathic effects of the vaccinia viruscan be reduced, higher level of the protein might be obtained. The findings provided a helpful means to furtherstudy the VP6 function and utilize the vaccinia virus/T7 RNA polymerase transient expression system.