Rodriguez J F, Smith G L
Department of Pathology, University of Cambridge, United Kingdom.
Virology. 1990 Jul;177(1):239-50. doi: 10.1016/0042-6822(90)90477-9.
A system for inducible gene expression by vaccinia virus (VV) vectors utilizing the Escherichia coli lac I repressor/operator system is described. A VV recombinant that expresses the lac I repressor protein from the constitutively active 7.5K promoter was constructed. Gel retardation experiments showed that a protein present in extracts of cells infected with this virus, but not wild-type virus, bound to the lac operator and that this binding was inhibited by IPTG. A series of VV recombinants were constructed that contained a 21-bp synthetic operator sequence(s) at different positions between the VV late 4b promoter and the firefly luciferase gene. Cells were co-infected with one of these viruses and the VV recombinant expressing the lac I repressor protein in the presence or absence of IPTG, and the level of luciferase activity was determined. Single operators positioned 19, 11, or 6 bp downstream of the promoter resulted in the 30, 50, or 97% inhibition of luciferase activity, respectively, while two operators increased the inhibition to greater than 99.9%. Addition of 1.25 mM IPTG at any time after infection restored 90% of enzyme activity from viruses containing a single operator, but reversal was only 50% when two operators were present. Both elements of the lac I inducible system were functional and stable in the genome of single recombinant virus. S1 nuclease protection of virus mRNA confirmed that luciferase expression was controlled at the transcriptional level and that IPTG did not affect transcription of endogenous VV genes. The utility of this inducible expression system for functional analyses of endogenous VV genes is demonstrated by the controlled expression of a gene encoding a 14-kDa protein and the correlation of 14-kDa expression with a biological property of the virus, namely plaque size phenotype. Plasmid vectors that are generally applicable to the inducible expression of genes by VV recombinants are described.
描述了一种利用大肠杆菌乳糖抑制蛋白/操纵子系统通过痘苗病毒（VV）载体进行诱导型基因表达的系统。构建了一种从组成型活性7.5K启动子表达乳糖抑制蛋白的VV重组体。凝胶阻滞实验表明，感染该病毒而非野生型病毒的细胞提取物中存在的一种蛋白质与乳糖操纵子结合，且这种结合被异丙基-β-D-硫代半乳糖苷（IPTG）抑制。构建了一系列VV重组体，它们在VV晚期4b启动子和萤火虫荧光素酶基因之间的不同位置含有一个21bp的合成操纵子序列。细胞在有或无IPTG的情况下与这些病毒之一和表达乳糖抑制蛋白的VV重组体共感染，并测定荧光素酶活性水平。位于启动子下游19、11或6bp处的单个操纵子分别导致荧光素酶活性抑制30%、50%或97%，而两个操纵子则将抑制作用提高到大于99.9%。感染后任何时间添加1.25mM IPTG可使含有单个操纵子的病毒恢复90%的酶活性，但当存在两个操纵子时，恢复率仅为50%。乳糖抑制诱导系统的两个元件在单个重组病毒基因组中均具有功能且稳定。对病毒mRNA进行S1核酸酶保护证实，荧光素酶表达在转录水平受到控制，且IPTG不影响内源性VV基因的转录。通过编码14kDa蛋白的基因的可控表达以及14kDa表达与病毒生物学特性即噬斑大小表型的相关性，证明了该诱导表达系统在内源性VV基因功能分析中的实用性。还描述了一般适用于通过VV重组体诱导基因表达的质粒载体。
