亚洲牛带绦虫泛素缀合酶E2的克隆表达及免疫鉴定

Cloning,expression and immunoassay of ubiquitin-conjugating enzyme E2(UBC E2)gene from Taenia asginata asiatica

目的克隆表达亚洲牛带绦虫泛素缀合酶E2(UBCE2)全长编码序列,获得纯化的重组蛋白,为进一步功能研究做充分准备。方法以亚洲牛带绦虫成虫cDNA文库中含有UBCE2基因的质粒作为模板,扩增该基因,将其克隆到原核表达载体pET-28a(+)中,测序鉴定重组质粒,在大肠杆菌BL-21/DE3中用IPTG诱导,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,并使用Western blotting分析其免疫反应性。结果PCR、双酶切及DNA测序结果均表明重组质粒pET-28a(+)-UBCE2构建成功,该基因在大肠杆菌中以包涵体形式得到表达,十二烷基肌氨酸钠纯化后蛋白通过SDS-PAGE鉴定结果表明该基因在大肠杆菌BL-21/DE3得到高效表达,亲和层析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患者血清识别,表明其有免疫反应性。结论亚洲牛带绦虫UBCE2可在原核表达系统中表达,并具有免疫反应性,为进一步研究该基因的功能奠定基础。

Objective To clone, express and conduct a preliminary immunoreactivity study on ubiquitinconjugating enzyme E2 gene from Taenia saginata asiatica, and obtain purified recombinant product to make full preparation for further function research. Methods The homologous sequence of ubiquitin-conjugating enzyme E2 （UBCE2） gene from adult Taenia saginata asiatica and its full-length coding region were obtained from the fulllength cDNA library by PCR and sequenced. Meanwhile, the encoding sequence was cloned into the prokaryotic expression vector pET-28a（＋）, then it was endonuclease digestion and was expressed in Escherichia colt BL21 with IPTG induction. The recombinant product was purified according to the protocol NTA agarose and detected by SDSPAGE, and the immunoreactivity study was conducted with Western blotting. Results PCR, double enzyme digestion and DNA sequencing results all indicated that pET-28a （＋） and UBCE2 recombinant plasmid was successfully constructed. SDS-PAGE results showed that gene expression took place in Escherichia colt BL-21/DE3, and highly purified protein was obtained after solution, refolding and particle exchange chromatography of inclusion body deposits. The recombinant protein reacted with the serum of patients infected by Taenia asiatica and Taeniarhynchus saginatus, which indicated its immunoreactivity. Conclusion The ubiquitin-conjugating enzyme E2 gene has been effectively expressed in prokaryotic expression system and its immunoreactivity has been confirmed, which provides favorable conditions for further studies of the gene.