T7RNA聚合酶真核表达质粒的构建及其病毒拯救功能的鉴定  

Construction and identification of a vector inserted with gene of T7 RNA Polymerase

作　　者：沈宏辉[1,2] 白冰珂[3] 柳昊东[4] 罗声栋[3] 胡燕[3] 侯俊[3] 王志杰[2] 孔维[1] 鲍一丹[5] 貌盼勇[2] 

机构地区：[1]吉林大学生命科学学院,长春130012 [2]解放军第三0二医院 [3]解放军第三0二医院实验技术研究保障中心 [4]桂林医学院生物技术学院 [5]中国疾病预防控制中心信息中心

出　　处：《中华实验和临床病毒学杂志》2011年第2期146-148,共3页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的建立TTRNA聚合酶真核表达质粒细胞内病毒拯救系统。方法利用分子生物学技术，以大肠埃希菌BL21（DE3）的T7RNA聚合酶基因为目的基因，构建TTRNA聚合酶真核表达质粒VR-1a并鉴定，然后将VR-1a和EV71病毒感染性克隆质粒共转染Vero细胞并传代，观察其细胞病变，同时用RT-PCR检测EV71病毒核酸和用ELISA检测EV71病毒抗原。结果酶切和测序显示成功构建T7RNA聚合酶真核表达质粒VR-1a，和EV71病毒感染性克隆共转染Vero细胞后出现明显的病变，RT．PCR检测EV71病毒核酸阳性并测序证实，ELISA检测显示有EV71病毒抗原。结论此方法可以用于细胞内拯救EV71病毒，有望应用于EV71病毒的核酸疫苗免疫研究。Objective To develop a system to rescue virus by intracellular expression of T7 RNA Polymerase. Methods The gene of T7 RNA Polymerase was amplified and cloned to VR1012 by molecular biological technology. The expression plasmid VR-la was then identified. VR-la and EV71 infectious plasmid were co-transfected in Vero cell. CPE was observed and viral gene viral antigen were detected. Results The gene of T7 RNA Polymerase was successfully cloned into vector VRI012. Vero cell developed to CPE after being transfected VR-la and EV71 infectious plasmid. EV71 gene was amplified by RT-PCR from the culture. EV71 antigen was also detected by ELISA. Conclusion The method can be used to rescue virus. It could aoolv to immunologic research of EV71 DNA vaccine.

关 键 词：DNA指导的RNA聚合酶 肠道病毒属 转染 

分 类 号：R3[医药卫生—基础医学]