SARS冠状病毒N蛋白激活hfgl2凝血酶原酶基因的研究  

Nucleocapsid Protein of SARS-associated Coronavirus Upregulates the Transcription of hfgl2 Prothrombinase /fibroleukin Gene

作　　者：姚华宁[1] 王战会[2] 韩梅芳[1] 侯金林[2] 罗小平[1] 宁琴[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院感染免疫研究室感染科,湖北武汉430030 [2]南方医院感染内科,广东广州510515

出　　处：《中国病毒学》2006年第4期319-323,共5页Virologica Sinica

基　　金：科技部973-SARS专项(2003CB514112);教育部首批SARS项目基金(教技司[2003]64号)

摘　　要：研究鉴定激活hfgl2凝血酶原酶基因的SARS冠状病毒结构蛋白。从SARS尸检肺组织中抽提RNA后制备cDNA,分别扩增SARS-CoV的N、S2和M全长基因序列,再分别克隆到真核表达载体pcDNA3.1(+)上。应用免疫组织化学分析鉴定pcDNA3.1-N、pcDNA3.1-M和pcDNA3.1-S2的表达。构建人纤维介素(hfgl2)启动子荧光素酶报告基因质粒,并将SARS冠状病毒结构蛋白表达质粒分别与其共转染以明确激活hfgl2基因转录的SARS冠状病毒结构蛋白。将目的片段克隆至pcDNA3.1(+),经酶切鉴定和测序鉴定无误;免疫组织化学染色可见明显的CHO细胞胞浆棕染。与hfgl2启动子共转染实验阐明SARS冠状病毒膜(M)蛋白和刺突糖(S2)蛋白对hfgl2基因的激活与对照组无显著差异,而SARS冠状病毒核心(N)蛋白可激活hfgl2启动子,使其转染活性提高4.6倍。SARS冠状病毒N蛋白可增强hfgl2基因的转录活性。The structural protein of SARS-associated coronavirus （SARS-CoV） responsible for the activation of hfgl2 gene was investigated. Gene fragments encoding the nucleocapsid, the membrane, and spike proteins were amplified by RT-PCR, and they were cloned into the eukaryotic expression vector pcDNA3.1 （＋）. The plasmids were verified by restriction endonuclease analysis and sequencing. Chinese hamster ovary （CHO） cells were co-transfected with a plasmid carrying the hfgl2 promoter/luciferase and with one of the recombinant plasmids containing the genes enconding the N, M and S structural proteins. Luciferase activity was assayed as a measure of promoter function. Immunohistochemistry showed that genes encoding the structural protein of SARS-CoV were successfully expressed only in those cells transfected with the recombinated plasmids. Co-transfection of N gene expression construct with the reporter construct containing hfgl2 promoter in CHO cells showed a remarkable increase in luciferase activity compared with nontransfected cells. However, there was no significant difference in luciferase activity in cells cotransfected with pcDNA3.1-M and with pcDNA3.1-S2 along with hfgl2 promoter/luciferase reporter gene. We conclude that the nucleocapsid protein of SARS-CoV upregulates the transcription of hfgl2 prothrombinase/fibroleuldn gene.

关 键 词：凝血致活酶 冠状病毒 核蛋白质类 基因表达调控 严重急性呼吸综合征 

分 类 号：R373[医药卫生—病原生物学]