汉滩病毒微基因组的构建与评价

Construction and evaluation of a minigenome system for Hantaan virus

目的建立汉滩病毒(Hantaan virus,HTNV)微基因组系统,并进行初步评价。方法用分子克隆方法构建HTNV微基因组系统所需的微基因组质粒和表达核衣壳蛋白(nucleocapsid protein,NP)、RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)的辅助质粒,转染HEK-293T细胞;并在辅助病毒添加与否的情况下进行验证。结果成功构建了基于HTNV L片段非编码区的绿色荧光蛋白(green fluorescence protein,GFP)和Gaussia荧光素酶(gaussia luciferase,Gluc)的微基因组质粒,GFP微基因组在辅助病毒HTNV感染的情况下可观察到明显绿色荧光,Gluc微基因组在辅助病毒感染或者辅助质粒共转染情况下均可检测到Gluc的表达。结论成功建立了HTNV的微基因组系统,为研究HTNV的复制机理和抗病毒药物的筛选提供了重要工具。

Objective To construct a minigenome system for Hantaan virus(HTNV),and to conduct a related preliminary evaluation.Methods The microgenomic plasmids required for the HTNV microgenome system and the helper plasmids expressing nucleocapsid protein(NP)and RNA polymerase(RNA-dependent RNA polymerase,RdRp)were constructed by molecular cloning method and transfected into HEK-293 T cells;and these were verified with or without helper virus.Results Both of the microgenomic plasmid based on green fluorescent protein(GFP)and Gaussia luciferase(Gluc)in the non-coding region of HTNV L fragment were successfully constructed.The GFP microgenome could be used in helper virus HTNV infection.The apparent green fluorescence was observed,and the expression of luciferase was detected in the Gluc microgenome in the case of helper virus infection or co-transfection of the helper plasmid.Conclusion The genomic system of HTNV has been successfully constructed,which provides an important tool for studying the replication mechanism of HTNV and screening of anti-viral drugs.