日本脑炎病毒(JEV)复制子表达载体的构建及其鉴定

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。

Construction and Application of Subgenomic Replicon Vectors of Japanese Encephalitis Virus

Based on the infectious clone of JEV vaccine SA14-14-2, the subgenomic replicons pCTCJEV, pCTMJEV 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.