Panicali D, Paoletti E
Proc Natl Acad Sci U S A. 1982 Aug;79(16):4927-31. doi: 10.1073/pnas.79.16.4927.
We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by cotransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.
我们构建了含有单纯疱疹病毒胸苷激酶基因的重组痘苗病毒。该基因被插入到已发生自发缺失的痘苗病毒变体的基因组中，以及大型原型痘苗病毒变体的120兆道尔顿基因组中。这是通过真核组织培养细胞与含有侧翼痘苗病毒DNA序列的单纯疱疹病毒克隆BamHI酶切胸苷激酶基因和感染性拯救痘苗病毒共转染，在体内进行重组来实现的。通过复制过滤技术或在生化选择培养基中培养重组病毒，获得了重组病毒的纯群体。作为插入痘苗病毒的单纯疱疹病毒胸苷激酶基因，在体内和体外均能转录，并且通过5-[125I]碘-2'-脱氧胞苷的磷酸化检测到体内转录为功能性基因产物的保真度。
