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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

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【作者】 唐辉滨何诚彭鲁英于伟英应其龙朱运松

【Author】 Tang Hui Bin He Cheng Peng Lu Ying Yu Wei Ying Ying Qi Long Zhu Yun Song (Department of Molecular Genetics,Shanghai Medical University,Shanghai 200032)

【机构】 上海医科大学基础医学院分子遗传学研究室

【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.

【Abstract】 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.

【基金】 国家自然科学基金
  • 【文献出处】 中国生物化学与分子生物学报 ,CHINESE JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY , 编辑部邮箱 ,1998年06期
  • 【分类号】Q784,
  • 【下载频次】90
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