PPARγ配体结合域的表达、纯化和鉴定  

THE EXPRESSION,PURIFUCATION AND DETECTION OF PPARγ-LIGAND BINDING DOMAIN

作　　者：王小行 周建 向珑 郭砚[2] 高小平 

机构地区：[1]成都地奥集团药物研究所,成都610041 [2]四川大学生命科学学院,成都610064

出　　处：《四川大学学报（自然科学版）》2001年第6期919-923,共5页Journal of Sichuan University(Natural Science Edition)

摘　　要：提取MCF 7乳腺癌细胞的总RNA ,通过RT PCR技术扩增出hPPARγ/LBDcDNA ,并克隆于载体pGEX 1γT上 ,测序表明该序列与已报道序列相同 ,将其转入BL2 1(DE3)大肠杆菌中 ,IPTG诱导表达GST hPPARγ/LBD ,在不同培养温度或IPTG诱导浓度下 ,GST hPPARγ/LBD以可溶或包涵体形式存在 ,表达产物经谷胱甘肽Sepharose 4B亲和层析纯化 ,可被抗hPPARγ多克隆抗体特异识别 。The hPPAR-LBD coding sequence was amplified with RT-PCR from the total RNA of human breast cancer cell line MCF-7,comfirmed by nucleotide sequencing, and then cloned into prokaryotic expression vector pGEX-1γT. The recombinant expression vector was transformed into E.coli strain BL21(DE3), and the expression of fusion protein GST-hPPAR-LBD was induced by IPTG. Under different culture temperatures or induced IPTG concentrations, the fusion protein was soluble or insoluble (inclusion bodies), and was purified by glutathione-Sephorose 4B affinity chromatography. The purified fusion protein bound its natural ligand specifically, and was also recognized by specific anti-hPPAR polyantibodies.

关 键 词：PPARγ配体结合域 基因表达 纯化 鉴定 抗糖尿病药物 乳腺癌细胞 克隆 

分 类 号：Q78[生物学—分子生物学] R977.15[医药卫生—药品]