[携带T7 RNA聚合酶基因的载体的构建与鉴定]
[Construction and identification of a vector inserted with gene of T7 RNA polymerase].
作者信息
Shen Hong-hui, Bai Bing-ke, Liu Hao-dong, Luo Sheng-dong, Hu Yan, Hou Jun, Wang Zhi-jie, Kong Wei, Bao Yi-dan, Mao Pan-yong
机构信息
College of Life Sciences, Jilin University, Changchun, 130012, China.
出版信息
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2011 Apr;25(2):146-8.
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-1a was then identified. VR-1a 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 VR1012. Vero cell developed to CPE after being transfected VR-1a 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 apply to immunologic research of EV71 DNA vaccine.
目的
通过T7 RNA聚合酶的细胞内表达来开发一种拯救病毒的系统。
方法
利用分子生物技术扩增T7 RNA聚合酶基因并将其克隆至VR1012。随后鉴定表达质粒VR-1a。将VR-1a与肠道病毒71型（EV71）感染性质粒共转染至Vero细胞。观察细胞病变效应（CPE）并检测病毒基因和病毒抗原。
结果
T7 RNA聚合酶基因成功克隆至载体VR1012。Vero细胞在转染VR-1a和EV71感染性质粒后出现CPE。通过逆转录聚合酶链反应（RT-PCR）从培养物中扩增出EV71基因。酶联免疫吸附测定（ELISA）也检测到了EV71抗原。
结论
该方法可用于拯救病毒。它可应用于EV71 DNA疫苗的免疫学研究。
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