日本脑炎病毒(JEV)复制子表达载体的构建及其鉴定  

Construction and Application of Subgenomic Replicon Vectors of Japanese Encephalitis Virus

作　　者：黄莺[1] 邵炜[1] 贾丽丽[2] 俞炜源[1] 俞永新[2] 

机构地区：[1]军事医学科学院生物工程研究所蛋白质工程室,北京100071 [2]中国药品生物制品检定所疫苗一室,北京100050

出　　处：《病毒学报》2007年第1期33-38,共6页Chinese Journal of Virology

摘　　要：在以往构建的JEV疫苗株SA14-14-2感染性克隆pBRkpn-JTF的基础上,构建了去除JEV结构区域基因(prM基因和E基因),保留非结构区域基因的复制子表达载体pCTCJEV、pCTMJEV,转染BHK-21细胞后,通过反转录PCR(RT-PCR)和间接免疫荧光实验(IFA)检测该载体的自主复制能力和蛋白表达情况,结果证实该载体在BHK-21细胞中能够自主复制并且表达非结构蛋白。进一步在该载体中插入报告基因EGFP,检测外源蛋白的表达情况,结果显示在转染复制子载体的24h后,荧光显微镜下能够看见绿色荧光蛋白的表达,阳性信号持续增强,能够维持10d左右,证实了构建的复制子载体pCTCJEV能够有效地表达外源蛋白,这为进一步建立以乙脑病毒复制子为基础的疫苗载体系统打下基础。Based on the infectious clone of JEV vaccine SA14-14-2, the subgenomic replicons pCTCJEV, pCTM-JEV with large deletions in the structural region were constructed. Then they were transfected into BHK-21 cell, the RNA replication of JEV subgenome can be monitored by RT-PCR and the non-structural protein can be found expressed in the cell by IFA. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive-strand and the negative-strand JEV RNA, we inserted an enhanced green fluorescence protein （EGFP） gene into the 3＇-UTR of pCTCJEV, pCTMJEV under the control of the internal ribosomal entry site （IRES） of encephalomyelocarditis virus RNA. After transfection, the EGFP fluorescence could be seen under the fluorescence microscope 1 day later, and maintained for more than a week with no apparent cytopathic effect. The constructed JEV replicons would provide valuable tools to provide a possible vector for a long-lasting RNA virus expression system.

关 键 词：日本脑炎病毒 复制子载体 外源蛋白 

分 类 号：R373.31[医药卫生—病原生物学]