亚洲牛带绦虫26kDa GST基因表达及免疫学分析

Cloning and prokaryotic expression of 26kDa glutathione S-transferase gene and recombinat protein immunogenicity analysis of Taenia saginata asiatica

目的对亚洲牛带绦虫成虫26kDa谷胱甘肽S-转移酶基因(glutathione S-transferase,GST)进行克隆、表达和免疫学初步分析研究,为深入研究其生物学功能提供依据。方法将亚洲牛带绦虫成虫26kDa GST克隆到原核表达质粒pET-30a(+)中,在大肠埃希菌BL-21/DE3中用异丙硫代-β-D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 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-30a(+)-TaGST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和亚洲牛带绦虫患者血清,表明其具有免疫反应性。结论亚洲牛带绦虫成虫26kDa谷胱甘肽S-转移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。

Objective： To clone and express the novel gene named glutathione S- transferase gene（GST） of Taenia saginata asiatica in order to analyze the immunogenicity of the recombinant protein and further research on the biological function of the gene. Methods By screening the full length cDNA plasmid library, the coding region of GST was amplified with PCR, and cloned into the prokaryotic expression vector pET - 30a （ ＋ ） 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. Analyze its immunogenicity by Western Blotting. Results PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was successfully constructed. The expression products were obtained and purified by Ni - IDA affinity chromatography. Western blot analysis of GST recombinant protein testified that the recombinant protein could be recognized by the serum of swine and patient immunized. GST recombinant protein also could ehcit efficiently specific antibodies in SD rats, therefore indicated its immunogenicity. Conclusion A novel gene coding GST of Taenia saginata asiatica was cloned, expressed, purified successfully. The purified protein of GST will be of importance for further research on the biological function of the gene.