表达人冠状病毒NL63棘突蛋白不同片段的重组痘苗病毒的制备与表达分析  

Characterization and Development of Recombinant Vaccinia Viruses Expressing Different Segments of Spike Protein Derived from Human Coronavirus NL-63

作　　者：赵国霞[1,2] 周为民[2] 陆柔剑[2] 王惠娟[2] 赵敏[1,2] 张庭瑛[1,2] 邓瑶[2] 高基民[1] 谭文杰[1,2] 

机构地区：[1]温州医学院医学病毒学研究所,温州325000 [2]中国疾病预防控制中心病毒病预防控制所病毒基因工程重点实验室,北京102206

出　　处：《病毒学报》2011年第3期250-256,共7页Chinese Journal of Virology

基　　金：国家863课题(2007AA02Z463;2007AA02Z464);传染病重大专项(2008zx10004-014);中澳卫生合作项目(CAHHF)EID07

摘　　要：HCoV-NL63是新近发现的人冠状病毒,对其外膜糖蛋白-棘突蛋白的表达及功能的研究仍有待深入。本研究利用天坛株痘苗病毒载体,克隆构建可表达HCoV-NL63棘突蛋白四个片段(N端棘突蛋白:S1;C端棘突蛋白:S2;受体结合区大片段:RL;受体结合区小片段:RS)的重组痘苗病毒(vJSC1175-S1;vJSC1175-S2;vJSC1175-RL;vJSC1175-RS),酶切测序证实表达载体构建正确,免疫荧光分析(IFA)各重组痘苗病毒中棘突蛋白不同片段的表达与定位,Western-Blot分析表明各种重组蛋白表达正确。分析结果显示:4种重组蛋白均能有效表达,S1、RL及RS蛋白的荧光主要分布在细胞膜上,而S2蛋白的荧光则主要分布于细胞浆,各个片段的分子量大小与文献报道相同,并可进行正确的翻译修饰(糖基化)。本研究首次采用痘苗病毒天坛株载体构建制备了表达HCoV-NL63棘突蛋白不同片段的重组痘苗病毒,为进一步分析人冠状病毒HCoV-NL63棘突蛋白的结构功能及探索其抗原性和免疫原性奠定了基础。The spike（S） glycoprotein of HCoV-NL63 is a major target in the development of diagnostic assays and vaccines,but its antigenic and immunogenic properties remain unclear.Four fragments coding spike proteins（S1,S2,RL and RS） from HCoV-NL63 were amplified and cloned into the expression vector derived from vaccinia virus（Tiantan strain）,and recombinant vaccinia viruses expressing four segments of spike proteins were generated（vJSC1175-S1;vJSC1175-S2;vJSC1175-RL;vJSC1175-RS）,respectively.Their expression location in cell and level were characterized using indirect immune fluorescence assay（IFA） and Western-Blot,respectively.The expressions of four segments of spike proteins in recombinant vaccinia viruses were showed at appropriate level and with posttranslational modification（glycosylation）,and S1,RL and RS were mainly distributed in the cell membrane,while the S2 was mainly distributed in the cytoplasm.Our results provide a basis for further exploring diagnostic role and vaccine development of different spike segments from HCoV-NL63.

关 键 词：人冠状病毒NL63 棘突蛋白 受体结合区 重组痘苗病毒 表达 

分 类 号：R373.1[医药卫生—病原生物学] Q78[医药卫生—基础医学]