REGULATED EXPRESSION OF FOREIGN GENES IN VACCINIA VIRUS UNDER THE CONTROL OF BACTERIOPHAGE-T7 RNA-POLYMERASE AND THE ESCHERICHIA-COLI LAC REPRESSOR
REGULATED EXPRESSION OF FOREIGN GENES IN VACCINIA VIRUS UNDER THE CONTROL OF BACTERIOPHAGE-T7 RNA-POLYMERASE AND THE ESCHERICHIA-COLI LAC REPRESSOR
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DOI：
10.1128/jvi.66.5.2934-2942.1992
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发表时间：
1992-05-01
影响因子：
5.4
通讯作者：
FUERST, TR
中科院分区：
文献类型：
--
作者：
ALEXANDER, WA;MOSS, B;FUERST, TR
将编码噬菌体T7 RNA聚合酶的基因（T7 gene 1）置于来自大肠杆菌lac操纵子的调控元件的控制下，构建完全由原核转录机器组成的诱导型痘苗病毒表达系统。 T7 RNA聚合酶的调节表达是构建稳定的含有T7启动子的重组牛痘病毒所必需的;否则，不受控制的表达导致干扰内源性病毒复制。 为此，将编码lac操纵子的阻遏蛋白的基因与病毒早期/晚期启动子融合，使其组成型表达，并将lac操纵子插入病毒主要晚期启动子和T7基因1之间。 获得了大于99%的T7 RNA聚合酶抑制，其在诱导剂异丙基-β-D-硫代半乳糖苷（IPTG）的存在下减轻约80倍。 将含有T7启动子控制的β-半乳糖苷酶报告基因的表达盒重组到含有T7基因1的病毒基因组的不同区域中。 分离出稳定的双重组病毒并使其生长至高滴度。 在没有诱导剂的情况下，β-半乳糖苷酶的表达基本上被抑制。 增加量的IPTG的添加诱导β-半乳糖苷酶的表达至抑制病毒复制的点。 这种混合的牛痘病毒系统（Vac/Op/T7）具有用于多种基因产物的有效生物生产的潜在应用。
The gene encoding bacteriophage T7 RNA polymerase (T7gene1) was placed under the control of regulatory elements from the Escherichia coli lac operon to construct an inducible vaccinia virus expression system consisting entirely of prokaryotic transcriptional machinery. Regulated expression of T7 RNA polymerase was necessary to construct a stable recombinant vaccinia virus harboring a T7 promoter; otherwise, uncontrolled expression led to interference with endogenous virus replication. To this end, the gene encoding the repressor protein of the lac operon was fused to a viral early/late promoter so that it was expressed constitutively, and the lac operator was interposed between a viral major late promoter and T7gene1. Greater than 99% repression of T7 RNA polymerase, which was relieved approximately 80-fold in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), was obtained. An expression cassette containing a T7 promoter-controlled beta-galactosidase reporter gene was recombined into a different region of the viral genome containing T7gene1. A stable, double recombinant virus was isolated and grown to a high titer. In the absence of inducer, beta-galactosidase expression was substantially repressed. Addition of increasing amounts of IPTG induced expression of beta-galactosidase to the point of suppression of viral replication. This hybrid vaccinia virus system (Vac/Op/T7) has potential applications for the efficient bioproduction of a wide variety of gene products.