应用RNA聚合酶Ⅰ反向遗传操作技术构建肾综合征出血热汉坦病毒微复制子  

Establishment of hemorrhagic fever with renal syndrome hantavirus minigenomes system by RNA polymerase I-driven system

作　　者：孙玉兰[1] 李川[2] 张全福[2] 李德新[2] 

机构地区：[1]北京北京市疾病预防控制中心传染病地方病控制所,100013 [2]中国疾病预防控制中心病毒病预防控制所

出　　处：《国际病毒学杂志》2013年第5期193-197,共5页International Journal of Virology

摘　　要：目的 构建肾综合征出血热病原汉坦病毒的微复制子,初步研究汉坦病毒基因组非编码区的调控功能.方法 利用RNA聚合酶Ⅰ体系,将报告基因绿色荧光蛋白（GFP）分别插入汉坦病毒76118毒株三个片段5＇和3＇非编码区之间,所形成的嵌合cDNA反向插入含RNA聚合酶Ⅰ的表达载体PHH21中,获得汉坦病毒三个片段的微复制子L-GFP-PHH21、M-GFP-PHH21、S-GFP-PHH21,将微复制子转染汉坦病毒76118株预先感染的vero细胞,48h后观察GFP表达情况.结果 汉坦病毒L、M、S三个片段微复制子均能观察到绿色荧光的表达,其中M片段最强,L片段最微弱.结论 以RNA聚合酶Ⅰ体系为基础构建的汉坦病毒L、M、S三个片段的微复制子是有功能的；汉坦病毒非编码区含有对汉坦病毒转录复制的重要调控原件.此微复制子系统可用于进一步研究汉坦病毒基因结构和功能的关系、基因转录和复制的调控机制,为实现汉坦病毒的病毒拯救奠定基础.Objective To develop minigenome system for hemorrhagic fever with renal syndrome hantavirus and to investigate the role of the noncoding regions of hantavirus in this process. Methods Com- plementary DNA （cDNA） containing the coding sequence for green fluorescence protein （GFP） was flanked by the 5＇ -and 3 ＇ -terminal untranslated regions of L, M, S segment of hantavirus 76118 strain. These chime- ric cDNAs （pol I expression cassette） were cloned into PHH21 vector which contain pol I promoter and ter- minator to generate artificial viral RNA genome segments （ minigenomes ）. These plasmids transfected into veto cells which has been infected by 76118 strain in advance and reporter gene activity was detected 24h N 48h post-transfection. Results Green fluorescence were observed for all the L, M and S segment-based minigenomes and the M segment-based minigenome showed the strongest level of GFP reporter. Conclusions We constructed a functional RNA polymerase I-driven minigenome system for hantavirus. The NCRs of hantavirus from all three segments contain the important regulation elements to initiate transcription. This minigenome system could be used as a powerful tool to study transcription and replication and constitute a valuable basis to rescue infectious hantavirus from eDNAs.

关 键 词：汉坦病毒 微复制子 RNA聚合酶Ⅰ 

分 类 号：R512.8[医药卫生—内科学]