乙脑病毒全长cDNA的扩增克隆及体外转录制备感染性RNA的研究  

Amplification and Cloning of Full-length JEV cDNA and Preparation of Infectious RNA by in vitro Transcription

作　　者：黄庆生[1] 马文煜[1] 姜绍谆[1] 樊英茹 张富泉[1] 肖毅[1] 丁天兵[1] 

机构地区：[1]第四军医大学微生物学教研室,西安710032

出　　处：《中国病毒学》2000年第4期330-335,共6页Virologica Sinica

摘　　要：本研究根据已知的乙脑病毒 (JEV)SA14株基因序列设计一套引物 ,利用长模板RT PCR技术 ,一次扩增出了JEV的全长cDNA ,并采用大片段克隆技术将其克隆于载体的RNA聚合酶启动子下游。阳性克隆转录的RNA转染HBK细胞后细胞产生病变 ,经乳鼠脑内接种及特异性免疫荧光染色证明为JEV ,这一结果表明JEV感染性克隆的构建获得了成功。其次 ,合成一条含有RNA聚合酶启动子序列的 5′引物 ,利用长模板RT PCR方法扩增出带有启动子的JEV全长cDNA ,以此cDNA为模板作体外转录。转录产物转染BHK细胞后观察到了典型的JEV病变。收获的病毒经乳鼠脑内接种及免疫荧光染色证明为JEV ,从而建立了制备JEV感染性RNA的快捷方法。本研究构建的JEV感染性克隆与建立的长模板RT PCR快速制备JEV感染性RNA的方法 ,将为JEV分子生物学研究的后续工作提供有力的工具和奠定坚实的基础。Preparation of infectious RNA by in vitro transcription from full length cDNA has led to advances in the study of many positive strand RNA viruses. There are two strategies of preparing full length cDNA of RNA viruses: (1)construction of infectious clone. (2)amplification of full length cDNA by long template RT PCR with the RNA promoter incorporated to 5′ primer. In this study both ways were established to generate Japanese Encephalitis Virus (JEV) infectious RNA. Firstly JEV (SA14 strain) full length cDNA was amplified by long template RT PCR and then cloned into the cosmid vector. One clone in which JEV cDNA was downstream of T7 promoter was selected and the RNA derived from this clone showed infectious after transfection of BHK cell. In another way of preparing JEV full length cDNA by long template RT PCR, T7 promoter was introduced to 5′ primer. RNA derived from this cDNA also showed infectious after transfection of BHK cell. The recovered viruses were further confirmed by intracerebral inoculation of newborn mice and immunofluorescent staining. These results suggested that JEV infectious clone was constructed and the long template RT PCR product directly used for in vitro transcription could provide a rapid and simple method for preparing JEV infectious RNA.

关 键 词：日本脑炎病毒 感染性RNA 体外不 全长CDNA 

分 类 号：Q785[生物学—分子生物学]