亚洲牛带绦虫磷脂酰肌醇转移蛋白α基因的克隆表达和免疫学分析  

Cloning and prokaryotic expression of phosphatidylinositol transfer protein alpha gene in Taenia saginata asiatica and the immunologic analysis of the recombinant protein

作　　者：吴璇[1] 黄江[1] 胡凤玉[2] 赵俊红[2] 胡旭初[2] 徐劲[2] 余新炳[2] 包怀恩[1] 

机构地区：[1]贵阳医学院寄生虫学教研室,贵阳550004 [2]中山大学中山医学院寄生虫学教研室,广州510080

出　　处：《中国人兽共患病学报》2008年第11期1022-1024,1028,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(30760227);贵州省科技攻关项目〔黔科合NY字(2006)3037〕联合资助

摘　　要：目的原核克隆表达亚洲牛带绦虫成虫磷脂酰肌醇转移蛋白α基因(Ta PITPα),探索其应用前景。方法将亚洲牛带绦虫成虫PITPα克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL-21/DE3中用IPTG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)进行鉴定,用镍离子金属螯合剂亲和层析柱进行纯化,纯化的重组蛋白用蛋白印迹(Western Blotting)进行免疫学分析。结果PCR、双酶切及DNA测序结果均表明pET-28a(+)-Ta PITPα重组质粒构建成功。SDS-PAGE结果表明目的基因在大肠埃希菌BL-21/DE3中获得高效表达,经亲和层析获得了高纯度蛋白。重组蛋白可被其免疫的SD大鼠血清和感染了亚洲牛带绦虫的猪血清识别,表明其具有免疫活性。结论亚洲牛带绦虫成虫磷脂酰肌醇转移蛋白α基因可在大肠埃希菌BL-21/DE3中获得具有免疫活性的高效表达,为进一步的功能研究奠定了基础。To clone and express the novel phosphatidylinositol transfer protein alpha gene in Taenia saginata asiatica （T. a. PITPa） in order to analyze the immunogenicity of the recombinant protein. By screening the full length cDNA plasmid library , the coding region of T. a. PITP a was amplified with PCR, and cloned into the prokaryotic expression vector pET-28a （ ＋ ） and then expressed in E. col i BL21 with IPTG induction. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography. And its immunogenicity was analysed by Western blotting. It was confirmed by PCR, double enzyme digestiom and DNA sequencing that the recombinant expression plasmid was successfully constructed. The expression products were obtained and purified by Ni-IDA affinity chromatography. Western blot analysis of T. a. PITP a recombinant protein testified that the recombinant protein could be recognized by immunized swine serum and could efficiently elicit specific antibodies in SD rats, indicating its immunogenicity. A novel gene coding PITP α of Taenia saginata asiatica was cloned, expressed,purified identification successfully. The purified protein of Ta PITP α will be of importance for further research on the biological function of this gene.

关 键 词：亚洲带绦虫 磷脂酰肌醇转移蛋白α 基因克隆 原核表达 

分 类 号：R383.3[医药卫生—医学寄生虫学]