汉坦病毒核心蛋白在大肠杆菌中的可溶性表达及鉴定  

Soluble expression of nucleocapsid protein from Hantavirus in E.coli and identification of its activity

作　　者：井申荣[1] 李光[1] 季秀玲[1] 王钰宁[1] 林连兵[1] 

机构地区：[1]昆明理工大学生物工程技术研究中心,云南昆明650224

出　　处：《云南大学学报（自然科学版）》2007年第S3期463-466,共4页Journal of Yunnan University(Natural Sciences Edition)

基　　金：国家自然科学基金资助项目(30570929);国家863计划重大资助项目(20060102A2212)

摘　　要：汉坦病毒是引起人类流行性出血热的病原体,为了获得具有细胞免疫的核心蛋白,采用PCR从S/pUC18载体上扩增含有较多CTL表达的汉坦病毒核心蛋白NP基因,通过TA克隆重组到T载体上,然后使用Nhe Ⅰ和Xho Ⅰ双酶切构建重组表达质粒NP/pET28a,鉴定正确后转化大肠杆菌BL21(DE3),不同温度下用IPTG诱导目的蛋白表达,SDS-PAGE和Western blot鉴定,结果表明获得了可溶性表达的核心蛋白,而且可与抗汉坦病毒的多克隆抗体呈阳性反应,为进一步研究核心蛋白的细胞免疫功能奠定了基础.Hantavirus is the major pathogen of epidemic hemorrhagic fever. In order to obtain its nucleocapsid protien (NP) with cellular immunity function, the truncated nucleocapsid protein gene contained many CTL epitope was amplified by PCR using recombinant plasmid S/pUC18 as template. The PCR product was inserted into T vector by TA cloning. The inserted NP sequence was digested with restriction enzyme Nhe I and Xho I,and then recloned into expression plasmid pET28a. The construct NP/pET28a was confirmed with enzyme digestion and DNA sequencing. E. coli BL21(DE3) was transformed with correct recombinant vector NP/pET28a. Then the engineering bacterium was induced for expression of NP with IPTG at different temperature. SDS - PAGE and Western blot were used to identification of the expressed protein. The results showed that nucleocapsid protein was successfully expressed at soluble form in E. coli and positively reactive with anti - hantavirus polyclonal antibody. This provides a basis for further study of the cellular immunity function of nucleocapsid protein.

关 键 词：汉坦病毒 核心蛋白 可溶性表达 

分 类 号：R378[医药卫生—病原生物学]