• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        A Simplified Method of Screening for Isolation of Recombinant Vaccinia Virus

        互联网

        995
        The utility of a recombinant vaccinia virus expression system for transient expression of genes was demonstrated in 1982 (1 ,2 ). Among a number of useful characteristics of this expression system are the capacity of the vaccinia virus genome to accommodate large genes (>20 kb pairs), availability of insertional sites on the viral genome yielding viable recombinant viruses, ability of the vaccinia virus to infect a wide range of mammalian hosts, cytoplasmic transcription and replication of the viral genome unlike many other expression systems, a relatively high level of recombinant proteins produced in infected cells, and the primary sequence-directed posttranslational modifications of recombinant proteins aim to native proteins (for a review, see ref. 3 ). As a first step in the construction of a recombinant vaccinia virus, the gene encoding a protein in the form of a cDNA is cloned into a plasmid vector under the control of an early or late vaccinia virus promoter (4 6 ), or a bacteriophage T7 promoter/the encephalomyocarditis virus 5′-untranslated leader sequence which would allow a cap independent mechanism of translation of the transcripts (7 ). Flanking the gene chosen for expression are DNA sequences representing portions of a nonessential viral gene such as that encoding the thymidine kinase in order to allow homologous recombination at the locus of the viral gene in vivo (6 ). The frequency with which the gene is integrated into the viral genome has been estimated to be about 0.1% (for a review, see ref. 3 ). In the original protocol for the isolation of a recombinant vaccinia virus (6 ), cell monolayers are infected with the wild-type virus (WR strain) and transfected with the plasmid DNA encoding the gene of interest. Since insertion of the gene of interest at the viral thymidine kinase (TK) gene locus results in recombinant viruses with the TK phenotype, plaque isolates of recombinant viruses are selected in the presence of 5-bromodeoxyuridine (5-BrdU). The conditions chosen for selection in the presence of this analog of thymidine are lethal to replication of the wild-type virus owing to incorporation of the phosphorylated form into the viral genome, but not to the recombinant virus deficient in TK. The cell lysates containing the wild-type and recombinant viruses at appropriate dilutions are used to infect cell monolayers of human TK cells, and then overlaid with growth medium containing agarose and 5-BrdU to prevent spreading of recombinant virus plaques and inhibit replication of the wild-type virus, respectively. A second agarose overlay containing the 5-BrdU and the neutral red stain is used to enable one to visualize recombinant virus plaques (6 ). A single plaque results from a productively infected cell and the cell-to-cell spread of the virus, and an agarose overlay localizes each of these plaques on a mono-layer. Since a plaque could consist of either a recombinant virus or a spontaneous TK mutant virus often present as a contaminant in the cell lysates, a suitable hybridization technique is employed in the screening protocol for isolation of recombinants (6 ).
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序