【摘要】 目的对亚洲牛带绦虫胞浆型苹果酸脱氢酶基因(malate dehydrogenase,MDH)进行克隆、表达和免疫学研究。方法将亚洲牛带绦虫成虫MDH克隆到原核表达质粒pET-30a(+)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-30a(+)-TaMDH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大鼠血清识别,表明其具有免疫原性;并且能识别感染了亚洲牛带绦虫的猪血清,表明其具有免疫反应性。结论亚洲牛带绦虫苹果酸脱氢酶基因可在原核表达系统中获得具有免疫学活性的高效表达,为进一步研究该蛋白的功能奠定了基础。更多还原

【Abstract】 To clone and express the movel gene named as malate dehydrogenase gene(MDH)in Taenia saginata asiatica in order to analyze the immunogenicity of its recombinant protein the coding region of MDH was amplified with PCR by screening the full length cDNA plasmid library,cloned into the prokaryotic expression vector pET-3a(+)and then expressed in E. coli BL21 with IPTG induction. The recombinant proteins expressed were identified by SDS-PAGE and purified by Ni-IDA affinity chromatography. In addition,the immunogenicity of the purified recombinant proteins was analyzed by Western blotting. As demonstrated by PCR,double enzyme digestion and DNA sequencing,it was confirmed that the recombinant expression plasmid was successively constructed. The MDH recombinant protein tested with Western blot analysis could be recognized by the specific immune serum from immunized SD rats and it could elect efficiently specific antibodies in SD rats,indicating the presence of its immunogenicity. It is apparent that the purified protein of MDH may be of importance for further studies on the biologic function of these proteins.更多还原