Application of poxvirus K3 ortholog as a positive selection marker for constructing recombinant vaccinia viruses with modified host range
Application of poxvirus K3 ortholog as a positive selection marker for constructing recombinant vaccinia viruses with modified host range
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DOI：
10.1016/j.mex.2020.100918
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发表时间：
2020-01-01
期刊：
影响因子：
1.9
通讯作者：
Deschambault, Yvon
中科院分区：
文献类型：
--
作者：
Cao, Jingxin;Layne, Christine;Deschambault, Yvon
牛痘病毒能够在广泛的脊椎动物细胞中复制。然而，两个病毒宿主范围基因E3 L和K3 L的缺失将使病毒在所有测试的细胞类型中的复制失败，除了具有缺陷PKR和RNase L活性的细胞。通过在E3 L和K3 L双基因敲除的痘苗病毒中表达不同的痘病毒K3同源蛋白，我们可以构建具有改变的宿主范围的突变型痘苗病毒。在这里，使用痘病毒K3直系同源物作为阳性选择标记，我们开发了一种概念验证方案来构建表达外源蛋白的重组牛痘病毒。与野生型牛痘病毒相比，这种重组病毒具有修饰的宿主范围。该方法具有以下优点：廉价：不需要额外的选择试剂;高效：几乎所有挑取的重组病毒噬斑都是表达目的蛋白的稳定重组病毒;快速：从重组DNA载体转染开始，一周内即可获得表达目的蛋白的纯化重组病毒。皇冠版权所有（C）2020由Elsevier B. V.出版
Vaccinia virus is capable of replicating in a wide range of vertebrate animal cells. However, deletion of the two virus host range genes, E3L and K3L, would render replication of the virus abortive in all the cell types tested, except the cells with defective PKR and RNase L activity. By expressing different poxvirus K3 ortholog proteins in the E3L and K3L double knockout vaccinia virus, we can construct a mutant vaccinia virus with modified host range. Here, using poxvirus K3 ortholog as a positive selection marker, we developed a proof-of-concept protocol to construct recombinant vaccinia viruses expressing foreign proteins. Such a recombinant virus has a modified host range in comparison to wild-type vaccinia virus. This protocol offers the following advantages:Cheap: no additional selection reagent is required.Highly efficient: nearly all recombinant virus plaques picked would be the stable recombinant virus expressing the protein of interest.Rapid: starting from transfection with the recombinant DNA vector, a purified recombinant virus expressing the protein of interest could be obtained within a week. Crown Copyright (C) 2020 Published by Elsevier B.V.