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

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．

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

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.