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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。
Fever with Thrombocytopenia Syndrome Bunyavirus (SFTSV) is a newly discovered Bunyavirus that causes severe fever in humans with thrombocytopenia syndrome. We use the RNA polymerase Ⅰ system, respectively, three fragments of SFTSV L, M, S micro-replicons to study its non-coding region regulatory function. The reporter gene GFP or Luciferase was inserted into the 5 ’and 3’ non-coding regions of SFTSV respectively, and the resulting chimeric cDNA was inserted into the expression vector containing RNA polymerase I pHH21, SFTSV microduplex recombinant plasmids L-GFP-pHH21, M-GFP-pHH21, S-GFP-pHH21, L-Luc-pHH21, M-Luc-pHH21 and S-Luc- 293T cells were co-transfected with VRTS-10 and NPV-1012-NP of SFTSV polymerase protein (L protein) and structural protein (N protein), and the expression of GFP was detected 24-48 hours later. Specific green fluorescence was observed in L, M, S fragment GFP micro-replicons. The quantitative results of luciferase showed that the expression levels of different regions of non-coding regions were different, suggesting that the non-coding regions of SFTSV could initiate transcription and replication of micro-replicons with different intensities.