An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA
An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA
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DOI：
10.1016/j.jviromet.2008.01.011
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发表时间：
2008-05-01
影响因子：
3.1
通讯作者：
Bird, R. Curtis
中科院分区：
文献类型：
--
作者：
Fan, Zhen-Chuan;Bird, R. Curtis
以低拷贝数质粒骨架构建的牛病毒性腹泻病毒（BVDV）全长cDNA克隆在细菌中繁殖时稳定性不佳。为提高稳定性，使用细菌人工染色体（BAC）质粒pBAS构建了基因组大小为12.3 kb的BVDV SD1株全长cDNA克隆。所得到的克隆pBSD1在两种不同培养温度下，于三种不同的大肠杆菌菌株中稳定繁殖至少10代，这通过对子代质粒测序得以确定。源自pBSD1的体外转录产物在大小上具有同源性，并且在牛肾细胞（MDBK）中感染效率高达约5.0×10⁵ 空斑形成单位/微克RNA。回收的病毒BSD1带有作为遗传标记的五个人工引入的沉默点突变，并且在病毒生长动力学、RNA复制和蛋白质表达方面与野生型SD1相似。这个BAC克隆为BVDV基因的操作和研究提供了一个稳定的反向遗传学系统。（c）2008爱思唯尔公司。版权所有。
Full length cDNA clones of bovine viral diarrhea virus (BVDV) with a low-copy number plasmid backbone have not proven to be stable when propagated in bacteria. To improve stability, pBAS, a bacterial artificial chromosome (BAC) plasmid was used to construct a full length cDNA clone of BVDV strain SD1, which has a genomic size of 12.3 kb. The resulting clone pBSD1 was propagated stably for at least 10 passages in three different Escherichia coli strains at two different incubation temperatures as determined by sequencing the progeny plasmids. In vitro transcripts derived from pBSD1 were homologous in size and had an infectious efficiency as high as similar to 5.0 x 10(5) FFU/mu g RNA in MDBK cells. The recovered virus, BSD1, harbored the five artificially introduced silent point mutations as genetic markers and was similar to wild type SD1 in viral growth kinetics, RNA replication, and protein expression. This BAC clone provides a stable reverse genetics system for manipulation and study of BVDV genes. (c) 2008 Elsevier B.V. All rights reserved.