发热伴血小板减少综合征布尼亚病毒微复制子的建立  

Establishment of Minireplicon System for Severe Fever with Thrombocytopenia Syndrome Bunyavirus

作　　者：于霞丽[1] 姜晓林[1,2] 王涛[1] 孙玉兰[1] 张硕[1] 李川[1] 张全福[1] 梁米芳[1] 毕振强[2,3] 李德新[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206 [2]山东大学,济南250012 [3]山东省疾病预防控制中心,济南250014

出　　处：《病毒学报》2012年第3期246-251,共6页Chinese Journal of Virology

基　　金：重大专项:重大传染病应急处置检测技术平台(2011ZX10004-001)

摘　　要：发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国新发现的一种布尼亚病毒,可引起人类严重发热伴血小板减少综合征。我们利用RNA聚合酶Ⅰ体系,分别构建SFTSV三个片段L、M、S微复制子,研究其非编码区调控功能。将报告基因绿色荧光蛋白(GFP)或荧光素酶(Luciferase)分别插入SFTSV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SFTSV微复制子重组质粒L-GFP-pHH21、M-GFP-pHH21、S-GFP-pHH21、L-Luc-pHH21、M-Luc-pHH21和S-Luc-pHH21,分别与成功表达SFTSV聚合酶蛋白(L蛋白)和结构蛋白(N蛋白)的质粒VR1012-L和VR-1012-NP共同转染293T细胞,24～48h后观察GFP表达情况或检测萤光素酶表达量。L、M、S片段GFP微复制子均可观察到特异性绿色荧光。荧光素酶定量结果显示其在不同节段非编码区中的表达量不同,提示SFTSV三个节段的非编码区启动微复制子转录和复制的强度不同。Severe fever with thromhocytopenia syndrome hunyavirus is a newly emerging virus in China, enveloped with a tripartite, single-stranded RNA genome of negative polarity. The regulatory elements for viral transcription and replication, as well as encapsidation and packaging signals, are thought to be located within these noncoding regions （NCRs）. The terminal nucleotides are genus specific and highly conserved. The funetion of the remaining nucleotides of the NCRs is still not well understood. In this study, we de- veloped the plasmid-driven RNA polymerase I minireplicon system for SFTSV firstly, using reporter genes GFP and lueiferase. The function of the noneoding regions of the three Bunyaviridae RNA segments （L, M, S） in transcription was analyzed. Reporter genes are successfully expressed in SFTSV minireplicon system. Our results suggest that the NCRs of SFTSV from all three segments contain the necessary sig- nals to initiate transcription. Quantitative detection of the lueiferase expression level shows that promoter activity in the three segments is different.

关 键 词：发热伴血小板减少综合征布尼亚病毒 反向遗传学 微复制子 报告基因 

分 类 号：R373.9[医药卫生—病原生物学] R558.2[医药卫生—基础医学]