猪带绦虫苹果酸脱氢酶基因的克隆表达及免疫学分析

Expression and purification of malate dehydrogenase gene in Taenia solium and immunologic analysis of the recombinant proteins

目的对猪带绦虫苹果酸脱氢酶基因(malate dehydrogenase,MDH)进行克隆,表达及免疫学特性的初步研究。方法将猪带绦虫MDH基因克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL21/DE3中用异丙基--βD-半乳糖苷(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 Blot)进行免疫学分析。结果成功构建pET-28a(+)-MDH重组质粒,并获得高纯度蛋白,该重组蛋白可被其免疫SD大鼠血清识别,同时也能被感染猪带绦虫的病人及猪、感染牛带绦虫病人及感染亚带绦虫病人血清所识别。结论猪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。

The objective of this study was to clone and express the gene named as malate dehydrogenase gene（MDH） in Taenia Solium,and to analyze the immunogenicity of its recombinant protein.The coding region of MDH was amplified with PCR,cloned into the prokaryotic expression vector pET-28a（＋） and expressed in E.coli BL21/DE3 with IPTG induction.In addition,the immunogenicity of the purified recombinant proteins was analyzed by Western blotting.PCR,double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was successfully constructed.The expression products were obtained and purified by His-Ni^2＋ affinity chromatography.Western blotting analysis of MDH recombinant protein testified that these proteins could be recognized by sera of the patients infected with T.asiatica and T.rhynchus saginatus.Results suggested that the MDH gene of T.solium has been cloned and expressed,and the purified protein has been confirmed with immunogenicity.