人细小病毒B19非结构基因ns1和7.5ku蛋白基因的克隆与表达  

Cloning and expression of nonstructural protein genes ns1 and 7.5 ku of Human parvovirus B19

作　　者：董衍明[1] 冯志鸿[1] 王也[1] 张楠[1] 王媛[1] 徐鹏[1] 李毅[1] 

机构地区：[1]华中师范大学生命科学学院,湖北武汉430079

出　　处：《中国病原生物学杂志》2010年第7期481-484,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30670081)

摘　　要：目的通过分子克隆技术,构建人细小病毒B19非结构基因ns1和7.5kugene的原核表达载体,并诱导重组NS1和7.5ku融合蛋白的表达,为蛋白的纯化奠定基础。方法以含有B19病毒全基因组感染性克隆为模板,用PCR法扩增目的基因ns1和7.5kugene,以pET-28a为表达载体,构建重组表达质粒pET28a-ns1和pET28a-7.5ku,转化E.coliBL21(DE3),获得重组工程菌株。经IPTG诱导培养6h后,通过SDS-PAGE、Western blot鉴定表达产物。结果成功构建了pET28a-ns1和pET28a-7.5ku,IPTG诱导能获得较高的目的蛋白。结论人细小病毒B19的非结构基因能在大肠埃希菌中获得成功的表达,为蛋白的纯化和抗体制备奠定基础。Objective Two recombinant expression vectors containing the ns1 and 7.5 ku genes of Parvovirus B19 were constructed to express NS1 and 7.5 ku fusion proteins in a prokaryotic expression system through induction of IPTG.Methods These two genes were amplified by PCR from the infectious clone of the full-length B19 genome and inserted into a pET28a expression vector to generate the recombinant plasmids pET28a-ns1 and pET28a-7.5 ku,respectively.The two plasmids were transformed into E.coli BL21 （DE3）,and then the fusion proteins were expressed by induction with IPTG and confirmed by SDS-PAGE and Western blotting.Results Prokaryotic expression vectors containing the ns1 and 7.5 ku genes were successfully constructed and corresponding fusion proteins were expressed at a very high level.Conclusion Nonstructural protein genes of human parvovirus B19 were expressed in E.coli,and corresponding antibodies were produced using target proteins after cleavage of fusion proteins by Factor-Xa.

关 键 词：人细小病毒B19 NS1 7.5ku基因 克隆与表达 

分 类 号：Q936[生物学—微生物学]