乙型脑炎减毒活疫苗株全长感染性克隆的构建及表达外源基因的初步研究  

Construction of a Full-length cDNA Clone of a Live Attenuated Vaccine Strain against Japanese Encephalitis Virus and Preliminary Study of Expressing Exogenous Gene

作　　者：胡兵[1] 杨爽[2] 方志正[2] 

机构地区：[1]湖北省疾病预防控制中心传染病防治研究所,武汉430079 [2]武汉生物制品研究所有限责任公司,武汉430060

出　　处：《病毒学报》2014年第6期652-660,共9页Chinese Journal of Virology

摘　　要：通过构建乙型脑炎病毒减毒株SA14-14-2的全长cDNA克隆,来初步探讨将其作为表达载体的可行性,为下一步利用乙型脑炎病毒作为载体构建嵌合病毒打下基础。利用长片段RT-PCR的方法分两段扩增出乙型脑炎病毒cDNA,通过片段两端的酶切位点,将cDNA依次连接到pACYC184载体。进一步利用分子克隆的手段,在乙型脑炎病毒基因组cDNA的3′非编码区插入增强型绿色荧光蛋白(Ehanced green fluorescent portein,EGFP)基因作为报告基因,通过体外转录和转染,拯救乙型脑炎病毒和表达绿色荧光蛋白的乙型脑炎嵌合病毒。采用RT-PCR、蚀斑实验和荧光显微镜观察等方法对恢复病毒进行鉴定。对恢复病毒进行连续6次细胞传代,从病毒生长特性和结构基因稳定性的角度对恢复病毒传代稳定性进行研究。结果表明成功地扩增并构建得到乙型脑炎病毒的全长cDNA,在此基础上进一步构建得到了乙型脑炎嵌合病毒rJEV-EGFP全长cDNA,经过体外转录和转染获得了活的rJEV病毒及嵌合病毒rJEV-EGFP,并采用了多种方法对其进行了鉴定,拯救出的恢复病毒在已传代的代次内稳定性良好,嵌合病毒rJEV-EGFP可稳定地表达绿色荧光蛋白。本研究应用反向遗传学技术构建并在BHK-21细胞中成功地拯救出了rJEV和rJEV-EGFP活病毒,同时也表明乙型脑炎病毒SA14-14-2株可以作为载体来表达外源基因。This study aimed to construct full-length cDNA clones of the Japanese encephalitis virus（JEV）SA14-14-2strain and discuss the feasibility of constructing chimeric viruses for exogenous gene expression based on the JEV genetic skeleton.Long-fragment RT-PCR techniques were applied to amplify JEV cDNAs,and two amplified fragments with corresponding restriction endonuclease sites at both ends were cloned into the pACYC184 vector sequentially.Using standard molecular techniques,the enhanced green fluorescent protein（EGFP）gene was inserted into the 3′non-coding region of JEV as a reporter gene.After in vitro transcription and transfection procedures,wild-type JEV and chimeric JEV that expressed the EGFP as the reporter gene were successfully rescued.The recovered viruses were characterized by RTPCR,plaque assays,and direct fluorescence microscopy.After six serial passage generations,the stability of the recovered viruses were studied in terms of virus growth characteristics and structural gene expression.The results showed that cDNA clones of rJEV and rJEV-EGFP were successfully constructed and rescued in BHK-21 cells after in vitro transcription and transfection.Each generation of the recovered viruses was stable and the chimeric virus rJEV-EGFP could stably express EGFP.The findings of this study indicate that both rJEV and rJEV-EGFP could be constructed and rescued in BHK-21 cells,and the JEV SA14-14-2strain could be obtained as a viral vector to express foreign genes.

关 键 词：乙型脑炎病毒 反向遗传学 全长cDNA感染性克隆 表达载体 增强型绿色荧光蛋白 

分 类 号：R373.9[医药卫生—病原生物学]