Blasco R, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Gene. 1995 Jun 9;158(2):157-62. doi: 10.1016/0378-1119(95)00149-z.
We developed a procedure for isolation of recombinant vaccinia viruses (re-VV) based solely on plaque formation, without a requirement for specific cell lines, selective medium or special staining. The system consists of two components: (i) a mutant non-plaque-forming VV and (ii) a plasmid vector that, through homologous recombination, can simultaneously introduce a foreign gene and repair mutation in the VV genome. The mutant VV contains a deletion of the vp37 gene, encoding a 37-kDa protein component of the viral outer envelope that is required for efficient viral spread on cell monolayers. The plasmid vector contains a functional vp37, a strong synthetic VV early/late promoter, unique restriction sites for gene insertion, and flanking segments of VV DNA for homologous recombination. Following infection and transfection of cells with the mutant VV and plasmid vector, respectively, re-VV are identified and isolated by their ability to form plaques. To evaluate the system, a re-VV that expresses the gene encoding influenza virus hemagglutinin (HA) was isolated simply by picking visible plaques.
我们开发了一种仅基于噬斑形成来分离重组痘苗病毒（re-VV）的方法，无需特定细胞系、选择性培养基或特殊染色。该系统由两个组件组成：（i）一种突变的非噬斑形成痘苗病毒和（ii）一个质粒载体，该质粒载体通过同源重组可同时在痘苗病毒基因组中引入外源基因并修复突变。突变的痘苗病毒缺失vp37基因，该基因编码病毒外膜的一种37 kDa蛋白质成分，是病毒在细胞单层上有效传播所必需的。质粒载体包含一个功能性vp37、一个强合成痘苗病毒早期/晚期启动子、用于基因插入的独特限制性酶切位点以及用于同源重组的痘苗病毒DNA侧翼片段。在用突变的痘苗病毒和质粒载体分别感染和转染细胞后，通过其形成噬斑的能力来鉴定和分离重组痘苗病毒。为了评估该系统，通过挑选可见噬斑简单地分离出了一种表达编码流感病毒血凝素（HA）基因的重组痘苗病毒。
