人白介素2受体γ链胞外区在巴斯德毕赤酵母中的表达  

Expression of Extracellular Domain of Human IL-2 Receptor γ Chain in Pichia pastoris GS115

作　　者：孙伟[1] 王富英[1] 周鹤峰[1] 刘银花[1] 胡亮 李官成[1] 

机构地区：[1]遵义医学院珠海校区,广东珠海519041 [2]浙江省湖州市吴兴区畜牧兽医局,浙江湖州313000

出　　处：《生物技术通讯》2010年第5期632-636,共5页Letters in Biotechnology

基　　金：广东省科技厅地市引导重点项目;珠海市科技课题(2004B36001086)

摘　　要：目的:在毕赤酵母GS115中表达重组人白细胞介素2受体γ链(rhsIL-2Rγ)胞外区。方法:用RT-PCR法从正常人淋巴细胞中获得IL-2Rγ胞外区基因;构建重组质粒pPIC9K-hsIL-2Rγ,用聚乙二醇法转入感受态GS115菌株,MD平板筛选His+转化子,用BMMY培养基诱导表达rhsIL-2Rγ;对重组蛋白进行免疫酶染色、SDS-PAGE及Western印迹鉴定。结果:克隆到目的片段,构建了重组质粒pPIC9K-hsIL-2Rγ;免疫酶染色、Western印迹等结果显示,重组质粒已成功转化GS115,并获得诱导表达的rhsIL-2Rγ。结论:在毕赤酵母GS115中表达了rhsIL-2Rγ,其蛋白条带有上移现象,分子较大,可能其糖基化过度或存在二聚体。Objective： To express the recombinant extracellular domain of human interleukin-2 receptor γ chain（rhsIL-2Rγ） in Pichia pastoris GS115.Methods： The gene coding extracellular domain of IL-2Rγ was obtained by RT-PCR from human lymphocytes,and was used to construct the expression plasmid pPIC9K-hsIL-2Rγ.The plasmid linearized by SacⅠ was transformed into GS115 with PEG method.Then the high-copy strains were induced in BMMY to express the interest protein.The recombinant protein was identified by using immunocytochemishry staining and Western blot.Results： The recombinant plasmid pPIC9K-hsIL-2Rγ was successfully constructed and transformed into GS115,and the rhsIL-2Rγ were expressed by inducing,checked with immunocytochemishry staining and Western blot.Conclusion： The rhsIL-2Rγ were successfully expressed in P.pastoris GS115,and the molecular weight of rhsIL-2Rγ protein showed a trend for large which may be attributive to potential glycosylation or existence of a form of dimmer.

关 键 词：白介素2受体γ链 胞外区 巴斯德毕赤酵母 分泌表达 

分 类 号：Q78[生物学—分子生物学]