猪带绦虫谷胱甘肽转移酶GST的原核表达及免疫学研究

Prokaryotic expression of glutathione transferase gene from Taenia solium and its immunological investigations

目的通过对猪带绦虫谷胱甘肽转移酶GST的表达,对其免疫性进行初步研究。方法利用生物信息学从猪带绦虫成虫cDNA质粒文库中筛选出谷胱甘肽转移酶(GST)基因,将其克隆到原核表达载体pET28a(+),经过双酶切、PCR鉴定后,异丙基-β-D半乳糖苷(IPTG)诱导表达,并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,重组后的蛋白用His-镍蛋白纯化柱纯化,纯化的重组蛋白用蛋白印迹进行免疫学分析,Western blotting鉴定该蛋白免疫反应性。结果成功构建了重组体,并得到高纯度蛋白,该蛋白可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。结论猪带绦虫谷胱甘肽转移酶可在原核系统中获得具有免疫反应性的高效表达。

In the present study, the prokaryotic expression of glutathione transferase （GST） gene from Taenia solium and its immunological properties were investigated by means of biological informatics methods. The GST gene from T. solium was screened from The eDNA plasmid library of the adult worms. This gene was cloned into prokaryotic expression plasmid pET28a（＋） and then expressed in E. coli BL21 （DE3） after double enzyme digestion, PCR identification and IPTG induction. The expressed product was identified by SD-PAGE and the recombinant protein was purified through purification column of His-Ni^2＋ protein. Meanwhile, the immunoreactivity of the purified protein was analyzed by Western blot assay. In these ways, the recombinants were successfully constructed and the highly purified proteins were obtained. It was demonstrated that these proteins could be recognized by sera of patients infected with T. asitica and T. rhynchus saginatus. From these observations, it is evident that highly efficient expression of GST of Taenia solium with definite immunoreactivity can be demonstrated in the prokaryotic expression system.