胶质细胞源性神经营养因子受体α1基因的克隆表达及其活性研究  

Cloning,Expression and Biological Activity Analysis of Rat GFRα1 Gene

作　　者：王丽梅[1] 陈哲宇[1] 朱伟[1] 张磬[2] 黄爱军[1] 路长林[1] 何成[1] 

机构地区：[1]第二军医大学神经生物学教研室,上海200433 [2]中国科学院上海生物化学与细胞生物学研究所,上海200031

出　　处：《生物化学与生物物理进展》2002年第5期771-775,共5页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金 (3 0 0 0 0 0 48) ;上海市青年科技启明星计划(0 1QB14 0 0 1) ;国家教育部优秀青年教师资助计划;国家重点基础研究"973" (19990 5 40 0 5 )资助项目~~

摘　　要：为了获得重组胶质细胞源性神经营养因子受体α1(glialcellline derivedneurotrophicfactorreceptoralpha1,GFRα1)并研究其生物学活性 ,从新生 4天的SD大鼠海马组织中提取总RNA ,通过RT PCR方法 ,扩增出GFRα1cDNA .将GFRα1cDNA克隆至含T7启动子的质粒 pET 2 8a (+)中 ,构建表达质粒 pET GFRα1,转化大肠杆菌BL2 1(DE3) ,获得表达菌株BLGFRα1.表达菌株经 1mmol/LIPTG诱导 3～ 5h后 ,GFRα1蛋白表达 ,并形成包涵体 .凝胶自动扫描分析表明 ,GFRα1表达量占全菌总蛋白的 2 1 5 % ,用Ni2 + NTA树脂纯化和复性后 ,纯度达 90 %以上 。To obtain recombinant glial cell line derived neurotrophic factor receptor alpha1 (GFRα1) and study its biological activity, the cDNA encoding the mature rat GFRα1 was isolated using RT PCR with total RNA extracted from newborn SD rat hippocampus tissue. The expression plasmid pET GFRα1 was constructed by inserting GFRα1 cDNA into plasmid pET 28a(+) containing T7 promoter and transformed into E.coli BL21(DE3).An expression strain BL GFRα1 was selected.SDS PAGE analysis revealed that the rat GFRα1 protein was highly expressed and accumulated up to 21 5% of the total bacterial proteins in the form of inclusion body after the induction. By Ni 2+ chelation affinity chromatography, up to 90% GFRα1 protein was purified. Purified and refolded GFRα1 protein could significantly mediate the ability of GDNF to promote the survival and induce the differentiation of PC12 cells.

关 键 词：胶质细胞源性神经营养因子受体α1 克隆 活性 基因表达 PC12细胞 

分 类 号：Q421[生物学—神经生物学] Q593.2[生物学—生理学]