中国仓鼠卵巢细胞中稳定表达发热伴血小板减少综合征病毒样颗粒新方法的研究  

New Method for Stable Expression of SFTS Virus-like Particles in CHO-K1 Cells

作　　者：李建东[1] 姜晓林[2] 张全福[1] 李川[1] 梁米芳[1] 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]山东省疾病预防控制中心,济南250014

出　　处：《病毒学报》2015年第3期245-250,共6页Chinese Journal of Virology

基　　金：传染病防治重大专项(2013ZX10004101;2011ZX10004-803-006)

摘　　要：为了探索发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)样颗粒的高效表达方法,本研究根据布尼亚病毒装配成熟的机制,建立SFTS病毒包膜糖蛋白和核蛋白融合表达质粒,并在表达载体中引入荧光蛋白报告基因,转染中国仓鼠卵巢细胞,传代培养两代后,根据荧光蛋白表达水平,采用有限稀释法,在荧光显微镜下,人工挑选细胞集落,建立病毒样颗粒稳定表达细胞系,并通过荧光蛋白表达水平的变化,评价细胞系的稳定性,采用间接免疫荧光、免疫印迹和电子显微镜对病毒样颗粒进行分析鉴定。结果显示,融合表达的包膜糖蛋白和核蛋白可在细胞内装配形成病毒样颗粒并分泌到胞外,在无选择压力条件下筛选的细胞系能够在较长时间内保持稳定,传代40次后报告基因表达水平无明显变化。本研究显示了通过病毒结构蛋白基因的修饰,可以改进病毒样颗粒制备方法,为相关生物制品的生产技术研究提供了重要线索。To explore a new method for stable expression of virus-like particles（VLPs）of the severe fever with thrombocytopenia syndrome（SFTS）virus,an expression plasmid for the membrane glycoprotein（GP）and nucleocapsid protein（NP）of the SFTS virus was constructed by fusion of the two proteins via a serine residue,and a yellow fluorescence protein（YFP）gene was introduced into the plasmid as a reporter.CHO-K1 cells were transfected with this plasmid,and stable cell lines constructed using the limited dilution method.Cellular colonies were hand-picked based on YFP with the help of fluorescence microscopy and expanded without selection pressure.Stability of cell lines was evaluated by monitoring of fluctuation of the intensity of YFP for 40 passages.VLP production was characterized using an indirect fluorescence assay,immunoblotting,and electronic microscopy.We showed that GP and NP fusion proteins could be assembled into VLPs in vivo,and that VLPs had similar morphologies to virus particles.Selected cell lines were stable for YFP expression：no significant fluctuation was detected in 40 passages.These data demonstrated the effectiveness of this new method for expression of structural proteins of the SFTS virus and screening for stable cell lines.Our results could provide new concepts for the production of biopharmaceuticals.

关 键 词：SFTS病毒 病毒样颗粒 转染 稳定表达 

分 类 号：R373.32[医药卫生—病原生物学]