土耳其斯坦东毕吸虫磷酸丙糖异构酶基因的克隆及在毕赤酵母中的表达

Cloning and expression of in Pichia pastoris triosephosphate isomerase gene of Orientobilharzia turkestanicum

利用RT-PCR技术从土耳其斯坦东毕吸虫(Orientobilharzia turkestanicum)成虫总RNA中扩增磷酸丙糖异构酶基因(TPI),鉴定后将目的片段与毕赤酵母表达载体pPIC9k连接,构建重组表达质粒pPIC9k-TPI,并将其电击转化到毕赤酵母GS115中,重组菌株经甲醇诱导后表达的TPI蛋白,经SDS-PAGE、western-blotting检测,并利用葡聚糖凝胶层析柱纯化。结果显示,成功地克隆了土耳其斯坦东毕吸虫TPI；重组毕赤酵母表达了分子质量为43 ku的TPI蛋白；葡聚糖凝胶层析过滤得到单一的TPI蛋白。

The full length triosephosphate isomerase gene was amplified from total RNA of Orientobilharzia turkestanicum by RT-PCR and the fragment was cloned into an expression vector pPICgk to construct recombinant plasmid pPIC9k-TPI. The Pichia pastoris GS115 cells were transformed with the recombinant plasmid by electroporation and the transformants were induced by methanol to express a protein of approximately 43 ku. The expressed protein was verified by Western-blotting and was purified by sephadex G-75 column. The protein was approximately 43 ku in size as determined by SDS-PAGE and it was confirmed to be triosephosphate isomerase by analysis using Western-blotting. The pure protein was gained by sephadex G-75 column. In conclusion, the high expression of the triosephosphate isomerase gene in Pichia pastoris GS115 cells would provide a candidate protein for the immune diagnosis and prevention of Orientobilharziasis.