亚洲牛带绦虫NOMO1基因在原核细胞中表达  

Cloning,prokaryotic expression and immunoreacticity of nodal modulator 1 gene of Taenia saginata asiatica

作　　者：廖兴江[1] 戴佳琳[1] 黄江[1] 胡旭初[2] 余新炳[2] 申萍香[1] 周灵贵[1] 郎书源[1] 

机构地区：[1]贵阳医学院多媒体形态学实验室,贵阳550004 [2]中山大学中山医学院人体寄生虫学教研室

出　　处：《中国公共卫生》2009年第5期555-557,共3页Chinese Journal of Public Health

基　　金：国家自然科学基金(30760227);贵州省科技攻关项目[黔科合NY字(2008)3060]

摘　　要：目的构建亚洲牛带绦虫NOMO1基因原核重组质粒,进行原核表达、纯化及免疫学研究。方法以亚洲牛带绦虫成虫cDNA文库中含NOMO1基因的质粒作为模板,扩增该基因,将其克隆到原核表达载体pET-28a(+)中,测序鉴定重组质粒后再行诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,并用蛋白免疫印迹(Western-blotting)分析其免疫反应性。结果成功建构了原核重组质粒pET28a(+)-NOMI1。该基因在大肠埃希菌中以包涵体形式存在,破包涵体后纯化蛋白通过SDS-PAGE鉴定结果表明该基因在大肠埃希菌BL-21/DE3得到高效表达。Western blotting结果显示,该重组蛋白可被亚洲牛带绦虫、牛带绦虫病人血清识别,具有免疫反应性。结论成功克隆亚洲牛带绦虫NOMO1基因、表达和纯化得到了该基因的重组蛋白并且证明该基因具有免疫反应性,为进一步研究该基因的功能提供条件。Objective To construct prokaryotic recombinant plasmids of nodal modulator 1 gene of Taenia saginata asitica, to express and purify the recombinant protein and to conduct preliminary immunoreacticity study. Methods A pair of primers was designed according to the known sequence of NOMO1 gene. The NOMO1 gene was amplified by PCR and the encoding sequence was cloned into the prokaryotic expression vector pET28a（ ＋ ） and then expressed in E. coli BL21 with IPTG induction. The recombinant protein was detected by SDS-PAGE. Protein and the immunoreacticity study was conducted with Westem-bloting. Results The recombinant plasmid pET28a（ ＋ ）-Ta NOMO1 was successfully constructed. SDS-PAGE results showed that the gene expression took place in Escherichia coli BL21/DE3, and highly pure protein was achieved after the dissolving, refolding and particle exchange chromatography of inclusion body deposits. Western blotting anlysis of NOMO1 recombinant protein testified that the recombinant protein reacted with Taenia asiatica and Taeniarhynchus saginatus infected patient serum, which indicated its immunoreacticity. Conclusion The NOMO1 gene is successfully cloned. The recombinant protein is obtained through expression and purification, and the gene＇ s immunoreacticity is confirmed, which provides basis for further studies of the gene.

关 键 词：亚洲牛带绦虫 NOMO1基因 基因克隆 免疫反应性 

分 类 号：R383.32[医药卫生—医学寄生虫学]