三种不同转染方法拯救病毒的比较研究  

Comparative study of three different transfection methods to rescue the virus

作　　者：侯俊 罗生栋 胡燕[2] 沈宏辉[2] 白冰珂[2] 柴燕涛[2] 王志杰[2] 貌盼勇[2] 

机构地区：[1]解放军医学院,北京100853 [2]解放军第三0二医院

出　　处：《中华实验和临床病毒学杂志》2013年第6期486-488,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金项目（81271846）

摘　　要：目的比较3种不同转染方法，探索最佳的拯救病毒策略。方法3种转染方法包括：①含T7RNA聚合酶启动子的全长肠道病毒71（EV71）感染性质粒（P—EV71）和T7RNA聚合酶真核表达质粒（VR-1a）共转染Vero细胞；②含T7RNA聚合酶启动子的EV71全长基因片段（PCR产物）与VR-1a共转染Vero细胞；③利用体外转录技术得到EV71病毒全长RNA，直接转染Vero细胞。分别观察3种方法转染后产生细胞病变效应（CPE）时间及病变情况，用RT—PCR扩增拯救病毒核酸，测序验证其正确性，用蛋白印迹方法检测拯救病毒抗原。结果3种方法均可成功转染Vero细胞得到拯救病毒，三种转染方法转染效果比较：方法③优于②、②优于①。结论三种转染方法均可成功拯救EV71病毒，以病毒RNA直接转染效果最好。Objective Comparing three different transfection methods to explore the best strategy of rescuing virus. Methods Three transfection methods include： （1） EV71 infectious plasmids including T7 RNA polymerase promote and T7 RNA polymerase eukaryotic expression plasmid were co-transfected in Vero cell. （2） Full-length PCR products of EV71 including T7 RNA polymerase promote and VR-la were co- transfected in Vero cell. （3） Virus RNA of EV71 was transcripted by T7 ribomaxTM epress large scale RNA production system was transfected in Vero cell directly. Respectively observe the time of producing CPE after transfecting. Viral gene and viral antigen were detected by RT-PCR and WB. Results Veto cell can be successfully transfeeted by three genetic patters. Transfection effect （3） is better than that of （2）, （2） is better than that of（1）. Conclusion Three genetic patters can be used to rescue virus. The best Effect was viral RNA transfection directly.

关 键 词：RNA 肠道病毒属 转染 

分 类 号：R440[医药卫生—诊断学]