亚洲牛带绦虫成虫延伸因子-1基因的表达、纯化及免疫学分析

目的 对亚洲牛带绦虫成虫延伸因子-1(elongation factor 1,EF-1)基因进行克隆、表达和免疫学初步研究.方法 将亚洲牛带绦虫成虫EF-1克隆到原核表达质粒pET-28a( )中,在大肠杆菌BL-21/DE3中用异丙硫代-β-D半乳糖苷诱导表达,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,用蛋白印迹法(Western blotting)进行免疫学分析.结果 PCR、双酶切及DNA测序结果 均表明重组质粒pET-28a( )-EF-1构建成功.重组蛋白可被感染了亚洲牛带绦虫患者血清和猪血清识别,表明其具有免疫反应性.结论 亚洲牛带绦虫成虫EF-1基因可在原核表达系统中获得具有免疫学活性的表达,为进一步研究该蛋白的功能奠定了基础.

Prokaryotic expression of elongation factor 1 gene of Taenia saginata asiatica and analysis of purification and immunogenicity of the recombinant protein

Objective To clone and express the novel gene named elongation factor 1 (EF-1) of Taenia saginata asiatica in order to analyze the immunogenicity of the recombinant protein.Methods By screening the full length cDNA plasmid library,the coding region of EF-1 was amplified with PCR,and cloned into the prokaryotic expression vector pET-28a(+) and then expressed in E.coli BL21 with IPTG induction.The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography,and its immunogenicity was analyzed by Western blotting.Results PCR,double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was successfully constructed.Western blot analysis of EF-1 recombinant protein testified that the recombinant protein could be recognized by immunizing the serum of swine and patient,therefore indicating its immunogenicity.Conclusion A novel gene coding EF-1 of Taenia saginata asiatica was cloned and expressed successfully.The purified protein of EF-1 will be of importance for further research on the biological function of the gene.