重组尿激酶型纤溶酶原激活物受体在酵母系统中的表达及鉴定  

Expression and Identification of Recombinant Urokinase type Plasminogen Activator Receptor in Pichia pastoris

作　　者：唐辉滨[1] 何诚[1] 彭鲁英[1] 于伟英[1] 应其龙[1] 朱运松[1] 

机构地区：[1]上海医科大学基础医学院分子遗传学研究室

出　　处：《中国生物化学与分子生物学报》1998年第6期673-679,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金

摘　　要：构建截断型可溶性尿激酶型纤溶酶原激活物受体（ｕ－ＰＡＲ）的原核及酵母表达质粒并分别在大肠杆菌和Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母中高效表达．利用ＰＣＲ扩增截断型可溶性ｕ－ＰＡＲｃＤＮＡ片段，并分别连入酵母及原核表达载体中，构建成表达质粒．后者经诱导表达的蛋白被用于免疫家兔，获得抗ｕ－ＰＡＲ的抗血清，用于对酵母表达产物的鉴定．前者在甲醇的诱导下表达，产物分泌至培养液中．结果表明，原核表达的ｕ－ＰＡＲ蛋白免疫家兔后获得高效价的抗血清，利用此抗血清所作Ｗｅｓｔｅｒｎｂｌｏｔ证实酵母表达蛋白具有ｕ－ＰＡＲ的免疫原性，表观分子量４０ｋＤ左右．Ｍｕｔ－表型在第５ｄ为最高（２．５μｇ／ｍｌ），Ｍｕｔ＋表型在第４ｄ为最高（７．５μｇ／ｍｌ）．因此，构建的可溶性ｕ－ＰＡＲ表达质粒在Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母细胞获得表达，为进一步竞争拮抗受体功能的研究奠定了基础．The expression plasmid of human soluble and truncated urokinase type plasminogen activator receptor (u PAR) was constructed and expressed in Pichia pastoris yeast cells and the u PAR was expected to be the scavenger of endogenous u PA and subsequently to inhibit the tumor cell metastasis.PCR method was used to amplify the soluble and truncated u PAR cDNA fragment and the u PAR expressing plasmids were constructed.Then they were expressed in the host of Pichia pastoris and in E.coli respectively.The u PAR protein expressed in E.coli was used to immunize the rabbit and finally got the antiserum against u PAR(1∶32).In yeast Pichia pastoris ,under the induction by methanol,the expressed u PAR was secreted into the medium at the level of 2 5～7 5 μg/ml.The expressed protein was confirmed by the antiserum of u PAR from the rabbit,which was approximately the molecular weight of 40 kD.In addition,the strains with Mut + phenotype secreted the maximum of u PAR appeared on the fourth day (7 5 μg/ml),while the maximum of u PAR with Mut - phenotype appeared on the fifth day (2 5 μg/ml).Therefore,it was concluded that the soluble and truncated u PAR was synthesized and secreted by Pichia pastoris ,which had laid the foundation for further studies on identifying the capacity of u PAR to inhibit the migration and invasion of tumor cells.

关 键 词：肿瘤 酵母 细菌侵染 μ-PAR 

分 类 号：Q513.2[生物学—生物化学] R730.2[医药卫生—肿瘤]