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目的对亚洲带绦虫真核翻译起始因子3(eukaryotic translation initiation factor3 subunit,EIFs 3)进行克隆及免疫学初步研究。方法利用在线生物信息学工具从亚洲带绦虫成虫cDNA文库中筛选出含有EIFs3基因,并将该基因克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL21/DE3中诱导表达,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,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(Western blotting)进行免疫学分析。结果 PCR,双酶切及DNA测序均显示重组体构建成功,用亲和层析法得到高纯度的蛋白,且该重组蛋白可被亚洲带绦虫及牛带绦虫及猪带病人血清识别,表明其具有免疫反应性。结论亚洲带绦虫成虫EIFs 33基因可在原核表达系统中获得具有免疫活性的高效表达。
Objective To clone and immunologically study the eukaryotic translation initiation factor 3 subunit (EIFs 3) of Taenia asiatica. Methods EIFs3 gene was isolated from the cDNA library of adult Taenia saginata by using online bioinformatics tools. The gene was cloned into prokaryotic expression vector pET-28a (+) and induced in Escherichia coli BL21 / DE3. The expressed product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the recombinant protein was purified with a His-Ni protein purification column. The purified recombinant protein was immunoblotted by Western blotting Analysis. Results PCR, double enzyme digestion and DNA sequencing showed that the recombinant was constructed successfully. The high purity protein was obtained by affinity chromatography, and the recombinant protein was recognized by the serum of tapeworm and Taenia saginata as well as the pigs. Is immunoreactive. Conclusion EIFs 33 gene of adult Taenia saginata could be expressed in prokaryotic expression system with high immunocompetence.