摘要
目的:构建携带绿色荧光报告基因的人冠状病毒OC43感染性克隆.方法:设计带有绿色荧光蛋白的人冠状病毒OC43感染性克隆基因组序列,分段合成后利用融合聚合酶链式反应等方法得到8个亚基因组片段,通过酵母转化关联重组技术获得重组质粒,转染HEK-293T细胞进行病毒拯救,收获转染细胞培养上清感染靶细胞分析病毒拯救情况.结果:获得人冠状病毒OC43感染性克隆重组质粒,将该质粒转染细胞后成功获得携带绿色荧光蛋白报告基因的人冠状病毒OC43重组病毒.结论:成功构建了人冠状病毒OC43感染性克隆并获得重组病毒,为针对冠状病毒的基础和应用研究提供了有效工具.
Abstract
Objective:To construct an infectious clone of HCoV-OC43 carrying the green fluorescent protein reporter gene.Methods:The whole genome sequence of the HCoV-OC43 infectious clone carrying the green fluorescent protein reporter gene was designed and synthesized into 32 segments.The segments were then assembled into 8 fragments by fusion PCR.Recombinant plasmids were obtained by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae.The recombinant plasmid was transfected into HEK-293T cells for virus rescue.The virus rescue was confirmed by infecting cells with the collected supernatants from transfected cells.Results:HCoV-OC43 recombinant virus carrying the green fluorescent protein was successfully rescued from infectious clone transfected cells.Conclusions:HCoV-OC43 infectious clone carrying the green fluorescent protein can be obtained using transfor-mation-associated recombination cloning method.The infectious clone provides an effective tool in basic and applied research on human coronaviruses.
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