Fuerst T R, Niles E G, Studier F W, Moss B
Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122-6. doi: 10.1073/pnas.83.21.8122.
DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.
将编码噬菌体T7 RNA聚合酶的DNA连接到痘苗病毒转录启动子上，并整合到痘苗病毒基因组中。重组痘苗病毒保留了感染性，并在哺乳动物细胞中稳定表达T7 RNA聚合酶。通过将编码β-半乳糖苷酶或氯霉素乙酰转移酶的DNA片段插入带有噬菌体T7启动子和终止子区域的质粒中构建靶基因。当细胞用重组痘苗病毒感染并转染含有靶基因的质粒时，后者会高水平表达。氯霉素乙酰转移酶活性比用劳斯肉瘤病毒长末端重复序列的增强子和启动子区域或猿猴病毒40早期区域调控的传统哺乳动物瞬时表达系统所观察到的活性高400 - 600倍。痘苗/T7杂交病毒构成了一个简单、快速、广泛适用且高效的哺乳动物表达系统的基础。
