乙型脑炎病毒表达载体的研究

Study on Japanese Encephalitis Virus Expression Vector

用RT-PCR方法分段扩增了乙型脑炎病毒SA14-14-2疫苗株5′、3′NCR,利用融合PCR技术在5′、3′NCR之间引入BamHⅠ酶切位点,将5′NCR置于T7启动子控制之下,构建乙脑病毒微复制子表达载体pMR。分别将绿色荧光蛋白(GFP)和汉滩病毒核蛋白编码区基因插入到pMR中,构建两种表达外源基因的乙脑病毒微复制子表达载体:pMR-GFP和pMR-84FliS。经荧光显微镜直接观察、Westernblot、ELISA等方法检测,证实外源基因能够在辅助病毒SA14-14-2感染的BHK-21细胞中表达。

Two fragments containing the 5 non-coding region (NCR) and the 3 NCR of Japanese encephalitis virus vaccine strain SA14-14-2 were produced by RT-PCR. A BamH restriction site was then introduced between the 5 NCR and the 3 NCR region by fusion PCR with the 5 NCR under the control of T7 promoter. The PCR product was then cloned into the pGEM-T Easy vector to obtain the minireplicon expression vector. The gene coding regions of green fluorescence protein (GFP) of plasmid pEFmyc ERGFP and nucleocapsid protein of hantaan virus strain 84Fli were inserted into the BamH site of the minireplicon expression vector between 5 NCR and 3 NCR region, respectively. It showed that the minireplicon could express the foreign genes in SA14-14-2 infected BHK-21 cells, which was tested by fluorescence microscopy, Western blot and ELISA. The 5,3NCR may play an important role during the replication, transcription, translation and viron packaging of SA14-14-2.