EPS8蛋白的表达、多克隆抗体制备及其亚细胞定位  

Subcellular Location of EPS8 by Its Expression and Preparation of Antiserum

作　　者：罗畅[1,2] 丁小凤[1,2,3] 孙一兵[1,2] 韩梅[1,2] 

机构地区：[1]湖南师范大学生命科学学院 [2]蛋白质化学与发育生物学教育部重点实验室 [3]南京大学模式动物研究所,中国南京210089

出　　处：《湖南师范大学自然科学学报》2008年第2期100-104,共5页Journal of Natural Science of Hunan Normal University

基　　金：国家973资助项目(2005CB522505);湖南省科技厅资助项目(05FJ4016)

摘　　要：EPS8(EGFR pathwaysub strate 8)是epidermal growth factor receptor(EGFR)重要的激酶活性底物之一,近年有研究表明其与神经胶质瘤密切相关.为了对该基因进行深入的功能研究,成功地将EPS8基因构建于原核表达载体pGEX-4T-2,转化BL21(DE3)获得融合表达产物,用GST-EPS8(-10-230aa)免疫新西兰大白兔获得兔抗EPS8多克隆抗体.采用Western Blot,用该抗体检测EPS8基因的真核表达产物,证明该抗体有较好的针对EPS8蛋白的专一性,可用于对EPS8的结构和功能研究.同时,用该抗体通过荧光免疫细胞对4种神经胶质瘤细胞系进行定位分析发现,EPS8蛋白主要分布于胶质瘤细胞的细胞质中.Eps8（EGFR pathway substrate 8）,originally identified as a substrate for the kinase activity of the epidermal growth factor receptor （EGFR）, studies in recent years showed that it is closely related with glioma. In order to study the expression,subeellular distribution and function of EPS8 protein,the coding region of the human EPS8 gene is successfully subeloned into the prokaryotie expression vector pGEX-4T-2. The vector is then introduced into E.eoli BL21 （DE3） cells for highly efficient expression. GST-EPS8 （-10-230） protein is used to immunize New Zealand white rabbit to produce polyelonal antibodies. The specificity of the anti-EPS8 antibody is successfully demonstrated by Western blot analysis on eukaryotie expressed products of EPS8 protein. Moreover,the EPS8 immunoreaetive glioma cells are found to be uniformly distributed in the cytoplasm.

关 键 词：EPS8 表达 抗EPS8多抗 亚细胞定位 

分 类 号：Q78[生物学—分子生物学]