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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 transient expression system of vaccinia virus / T7 RNA polymerase has been widely used to express foreign proteins due to its many advantages over other expression systems. The VP6 DNA encoding fragment of TB-Chen strain rotavirus was inserted between the bacteriophage T7 promoter and terminator of prokaryotic plasmid pETL to obtain the recombinant expression plasmid pET-VP6. The constructed recombinant plasmid pET-VP6 was transfected into eukaryotic cell line MA104 by lipofectamine and re-infected with recombinant vaccinia virus vTF7-3 carrying the phage T7 RNA polymerase gene to detect the expression of the target protein VP6 in the cell. The results also show that vaccinia virus vTF7-3 infection, the cytopathic effect quickly, seriously affecting the protein expression. The results suggest that the protein may be highly expressed if it reduces the cytopathic effects of vaccinia virus vTF7-3 without affecting its ability to synthesize T7 RNA polymerase in cells. Provide theoretical basis for further study of the characteristics of VP6 gene and better application of vaccinia virus / T7 RNA polymerase transient expression system.