A cosmid-based system for inserting mutations and foreign genes into the simian varicella virus genome
A cosmid-based system for inserting mutations and foreign genes into the simian varicella virus genome
复制标题
DOI：
10.1016/j.jviromet.2005.06.013
复制
发表时间：
2005-12-01
影响因子：
3.1
通讯作者：
Mahalingam, R
中科院分区：
文献类型：
--
作者：
Gray, WL;Mahalingam, R
猿猴水痘是一种非人类灵长类动物的天然水痘样疾病。猴水痘病毒（SVV）与水痘-带状疱疹病毒（VZV）有遗传关系，非人灵长类动物感染水痘-带状疱疹病毒是研究水痘-带状疱疹发病机制和潜伏期的有效模型。在这项研究中，我们报告了一个基于宇宙的遗传系统的发展，以产生SVV突变病毒。将跨越病毒基因组的SVV亚基因组DNA片段（32- 38kb）克隆到cosmid载体中。用代表SVV整个基因组的四个重叠的cosmid克隆共转染Vero细胞，导致重组和感染性病毒的产生。SVV突变体是通过操纵一个cosmid并将其替换到遗传系统中产生的。这种遗传方法被用于在SVV开放阅读框14中插入一个位点特异性突变，该突变编码非必需糖蛋白C基因。在随后的实验中，将绿色荧光蛋白（GFP）基因插入到orf14内的SVV基因组中。这些SVV突变体在细胞培养中与野生型S 'VV一样有效地复制。该基因系统将有助于研究病毒突变对SVV发病机制和潜伏期的影响，也有助于开发和评价表达外源抗原的重组水痘疫苗。(c) 2005 Elsevier B.V.版权所有
Simian varicella is a natural varicella-like disease of nonhuman primates. The etiologic agent, simian varicella virus (SVV), is genetically related to varicella-zoster virus (VZV) and SVV infection of nonhuman primates is a useful model to investigate VZV pathogenesis and latency. In this study, we report development of a cosmid-based genetic system to generate SVV mutant viruses. SVV subgenomic DNA fragments (32-38 kb) that span the viral genome were cloned into cosmid vectors. Co-transfection of Vero cells with four overlapping cosmid clones representing the entire SVV genome resulted in recombination and generation of infectious virus. SVV mutants were produced by manipulation of one cosmid and substitution into the genetic system. This genetic approach was used to insert a site-specific mutation within the SVV open reading frame 14 which encodes the nonessential glycoprotein C gene. In a subsequent experiment, the green fluorescent protein (GFP) gene was inserted into the SVV genome within ORF 14. These SVV mutants replicate as efficiently as wild-type S`VV in cell culture. This cosmid-based genetic system will be useful to investigate the effect of viral mutations on SVV pathogenesis and latency and also to develop and evaluate recombinant varicella vaccines that express foreign antigens. (c) 2005 Elsevier B.V. All rights reserved.