淮阳山病毒NSs基因在THP-1细胞中的稳定表达及其对白细胞介素-6的调节作用  

Stable Expression of Huaiyangshan Virus NSs Protein in THP-1 Cells and Its Regulating Effect on IL-6

作　　者：戴晓懿[1] 郑雅匀[1] 金东[1] 孙晖[1] 罗雪莲[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《氨基酸和生物资源》2016年第2期23-27,共5页Amino Acids & Biotic Resources

基　　金：病毒学国家重点实验室开放课题(2015KF003);国家重大传染病防治科技重大专项(2013ZX10004-101)

摘　　要：淮阳山病毒(Huaiyangshan virus,HYSV)是一种能引起人类发热伴血小板减少综合征的新型布尼亚病毒。NSs是淮阳山病毒的毒力因子,可能在病毒的致病中起重要的作用。本研究从构建好的含HYSV NSs基因的重组质粒pBluntNSs中扩增出NSs基因,然后克隆到慢病毒载体pHAGE-CMV-MCS-IZsGreen中构建重组质粒pHAGE-NSs;利用脂质体将pHAGE-NSs与包装质粒psPAX2、包膜质粒pMD2.G共转染293T细胞,在荧光显微镜下可见大量绿色荧光,收上清,即慢病毒悬液;将慢病毒悬液感染THP-1细胞后,通过绿色荧光蛋白进行流式分选、Western blot验证获得稳定表达NSs蛋白的THP-1细胞。利用人白介素-6ELISA检测试剂盒检测NSs蛋白对白介素-6的调节作用,结果表明,NSs蛋白能激活THP-1细胞产生白细胞介素-6。Huaiyangshan virus（HYSV）is a novel bunyavirus,causing fever with thrombocytopenia syndrome.NSs was virulent factor in HYSV,and may play an important role in the pathogenesis of HYSV.NSs gene was amplified from the constructed plasmid pBlunt-NSs and then cloned into the lentiviral vector pHAGE-CMV-MCS-IZsGreen.Using lipofectamine 2000,the recombinant plasmid pHAGE-NSs,the packaging plasmid psPAX2 and the envelope plasmid pMD2.G were co-transfected into 293 Tcells.Fluorescent NSs protein in 293 Tcells was visualized using fluorensent microscopy.The supernatant of lentivirus was harvested,and then infected THP-1cells.THP-1cells stably expressing HYSV NSs protein were obtained by flow cytometry assay（FCM）screening and the expression of NSs protein was verified by Western blot.Using human interleukin 6ELISA kit,the role of NSs protein for regulation of interleukin 6was detected.The results showed that NSs protein could stimulate the expression of interleukin 6in THP-1cells.

关 键 词：淮阳山病毒 NSs基因 慢病毒载体 白介素6 

分 类 号：R373.3[医药卫生—病原生物学]