GENERAL-METHOD FOR PRODUCTION AND SELECTION OF INFECTIOUS VACCINIA VIRUS RECOMBINANTS EXPRESSING FOREIGN GENES
GENERAL-METHOD FOR PRODUCTION AND SELECTION OF INFECTIOUS VACCINIA VIRUS RECOMBINANTS EXPRESSING FOREIGN GENES
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DOI：
10.1128/jvi.49.3.857-864.1984
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发表时间：
1984-01-01
影响因子：
5.4
通讯作者：
MOSS, B
中科院分区：
文献类型：
--
作者：
MACKETT, M;SMITH, GL;MOSS, B
质粒载体的构建为表达外源基因的传染性痘苗病毒重组体的制备和筛选提供了便利。这些载体包含牛痘病毒胸苷激酶（TK）基因的全部或部分，由位于TK启动子附近的多个独特的限制性内切酶位点或TK基因内易位的另一个启动子中断。在一个独特的限制性内切酶位点插入外源蛋白的连续编码序列，将牛痘启动子的转录起始位点和外源基因的翻译起始位点并置。用这种质粒转染牛痘病毒感染的细胞后，嵌合基因两侧的牛痘病毒序列与病毒基因组内的相同序列之间发生同源重组。以这种方式形成的重组体在牛痘病毒启动子的控制下，将嵌合基因插入牛痘病毒TK基因体内。由于重组体有一个中断的TK基因，因此根据其TK-表型选择重组体，然后检查外源基因的存在和表达。构建表达原核酶氯霉素乙酰转移酶的感染性重组病毒，对系统进行优化。在未感染的细胞或被野生型牛痘病毒感染的细胞中，氯霉素乙酰转移酶活性的缺失，以及一种敏感和定量的酶测定的可用性，使人们能够估计各种启动子结构的相对强度。在重组病毒感染细胞后1 h内检测到氯霉素乙酰转移酶的表达，反映了所使用的启动子的早期性质。
The production and selection of infectious vaccinia virus recombinants expressing foreign genes was facilitated by the construction of plasmid vectors. These vectors contain all or part of the vaccinia virus thymidine kinase (TK) gene interrupted by multiple unique restriction endonuclease sites placed adjacent to the TK promoter or another promoter translocated within the TK gene. The insertion of a continuous coding sequence for a foreign protein at one of the unique restriction endonuclease sites juxtaposes the transcriptional start site of a vaccinia promoter and the translational start site of a foreign gene. After transfection of vaccinia virus-infected cells with such plasmids, homologous recombination occurs between the vaccinia virus sequences flanking the chimeric gene and the same sequences within the virus genome. Recombinants formed in this manner have the chimeric gene inserted within the body of the vaccinia virus TK gene under control of a vaccinia virus promoter. Since recombinants have an interrupted TK gene, they are selected on the basis of their TK- phenotype and then checked for the presence and expression of the foreign gene. Infectious recombinant viruses expressing the prokaryotic enzyme chloramphenicol acetyltransferase were constructed to optimize the system. The absence of chloramphenicol acetyltransferase activity in uninfected cells or in cells infected with wild-type vaccinia virus and the availability of a sensitive and quantitative enzyme assay allowed an estimation of the relative strengths of various promoter constructs. The expression of chloramphenicol acetyltransferase was detected within 1 h after infection of cells with recombinant virus, reflecting the early nature of the promoters used.