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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
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魏佳1,2,徐梅倩2,何国声2 ,姚宝安1
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（1.华中农业大学 动物医学院，湖北 武汉 430070； 2.中国农业科学院 上海兽医研究所，上海 200232）
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摘要：
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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43ku的TPI蛋白；葡聚糖凝胶层析过滤得到单一的TPI蛋白。 |
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关键词：
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土耳其斯坦东毕吸虫；磷酸丙糖异构酶基因；基因克隆；巴斯德毕赤酵母；表达 |
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中图分类号：S 852.735
文献标识码：
A 文章编号：1673-4696(2006)08-0634-05 |
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Cloning and expression of in Pichia pastoris triosephosphate
isomerase gene of Orientobilharzia turkestanicum |
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WEI Jia1,2 ,XU Mei-qian2,HE
Guo-sheng2,YAO Bao-an1 |
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（1.College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;
2.Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200232,China) |
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Abstract: 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 pPIC9k 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 43ku.The expressed protein was verified by Western-blotting and was purified by sephadex G-75 column.The protein was approximately 43ku 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. |
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Key words: Orientobilharzia turkestanicum；triosephosphate isomerase；gene cloning； Pichia pastoris；expression |
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