SELECTION OF RECOMBINANT VACCINIA VIRUSES ON THE BASIS OF PLAQUE-FORMATION
SELECTION OF RECOMBINANT VACCINIA VIRUSES ON THE BASIS OF PLAQUE-FORMATION
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DOI：
10.1016/0378-1119(95)00149-z
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发表时间：
1995-06-09
期刊：
影响因子：
3.5
通讯作者：
MOSS, B
中科院分区：
文献类型：
--
作者：
BLASCO, R;MOSS, B
我们开发了一种分离重组牛痘病毒（re-VV）的方法，该方法仅基于空斑形成，而不需要特定的细胞系、选择性培养基或特殊染色。该系统由两个组件组成：（i）突变的非空斑形成VV和（ii）质粒载体，通过同源重组，可以同时引入外源基因并修复VV基因组中的突变。突变VV含有缺失的vp 37基因，编码病毒外膜的37 kDa蛋白组分，该蛋白组分是病毒在细胞单层上有效传播所需的。质粒载体含有功能性vp 37、强合成VV早期/晚期启动子、用于基因插入的独特限制性位点和用于同源重组的VV DNA侧翼片段。分别用突变VV和质粒载体感染和转染细胞后，通过它们形成噬斑的能力鉴定和分离re-VV。为了评价该系统，通过简单地挑选可见的噬斑来分离表达编码流感病毒血凝素（HA）的基因的re-VV。
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 the 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.