亚洲带绦虫60S核糖体蛋白L8基因的克隆表达和免疫学分析

Cloning and prokaryotic expression of 60S ribosomal protein L8 gene of Taenia saginata asiatica and analysis on immunogenicity of the recombinant protein

目的原核克隆表达亚洲带绦虫成虫60S核糖体蛋白L8基因(TaRPL8),探索其应用前景。方法用RT-PCR方法从亚洲带绦虫成虫cDNA中获取RPL8基因,克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL21/DE3中用IPTG诱导表达,表达产物通过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进行免疫学分析。结果 PCR、双酶切及DNA测序结果均表明pET-28a(+)-TaRPL8重组质粒构建成功。SDS-PAGE结果表明目的基因在大肠埃希菌BL21/DE3中获得高效表达,亲和层析获得了高纯度蛋白。重组蛋白可被其免疫的SD大鼠血清识别但不能够被正常大鼠血清识别,表明其具有免疫原性。结论亚洲带绦虫成虫TaRPL8基因可在大肠埃希菌BL21/DE3中获得具有免疫原性的表达,为进一步的功能研究奠定了基础。

To clone and express the novel gene named 60S ribosomal protein 1.8 gene of Taenia saginata asiatica （Ta RPL8） in order to analyze the immunogenicity of the recombinant protein. The full-length cDNA of RPL8 gene was cloned from Taenia saginata asiatica using RT-PCR; and cloned into the prokaryotic expression vector pET-28a （ -F ） and then expressed in E. col i BL21 with IPTG induction. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography. And its immu- nogenicity was analyzed by Western Blotting. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant ex- pression plasmid was successfully constructed. The expression products were obtained and purified by Ni-IDA affinity chromatography. Western blot analysis of T.a. RPL8 recombinant protein testified that it could be recognized by immunized SD rat serum but couldn＇t be recognized by normal SD rat, which indicated its immunogenicity. Ifs suggested that a novel gene coding RPL8 of Taenia saginata asiatica was cloned, expressed, purified and identified successfully. The purified protein of Ta RPL8 coule be of importance for further research on the biological function of the gene.