重组人截短型IL-6表达载体的构建及表达  

Construction of truncated human IL-6 recombinant and its expression

作　　者：孔令洪[1] 王一理[1] 韩俊宏[1] 郑瑾[1] 齐旭[1] 耿宜平[1] 司履生[1] 

机构地区：[1]西安交通大学医学院,陕西西安710061

出　　处：《西北大学学报（自然科学版）》2001年第2期149-152,共4页Journal of Northwest University（Natural Science Edition）

基　　金：美国中华医学基金!资助项目 (CMB92 - 5 5 0 )

摘　　要：旨在建立人截短型 IL- 6原核高效表达系统。用 PCR方法获得截短的人 IL- 6基因 ( rh IL - 6c DNA) ,将其插入克隆载体 p UC1 8中 ,经证实后再克隆入表达载体 p BV2 2 0中 ,鉴定阳性表达载体。结果建立了截短型 IL- 6基因的克隆载体 ,经测序证实完全正确 ;构建了截短型 IL- 6的原核表达质粒 p BV2 2 0 /rh IL- 6并获得高效表达 ;初步纯化的表达蛋白的比活性为 5× 1 0 7μmol/min· mg蛋白 ;用基因工程技术在大肠杆菌中高效表达了截短型 rh IL- 6,为大量制备 IL- 6奠定了基础。To construct truncated human IL 6 cDNA expression recombinant and express the truncated IL 6 protein in E. coli DH5α. The truncated IL 6 gene was cloned into cloning vector pUC18 after being amplified by PCR, and the sequence was confirmed by DNA sequencing,restriction enzyme digestion and PCR identification. Then, the truncated IL 6 cDNA fragment was inserted into expression plasmid pBV220, and pBV220/hIL 6 was transformed into E. coli DH5α. Cloning vector containing truncated hIL 6 was constructed and confirmed correct by DNA sequencing; a prokaryotic expression recombinant pBV220/hIL 6 was constructed and engineering DH5α was obtained. A high level expression of truncated IL 6 protein which accounted for about 32% of the total cellular protein was observed from SDS PAGE analysis after heat induction. And cell culture experiment showed that crudely extracted IL 6 protein had a specific activity of 5×10 7 μmol/min·mg protein. A high expression syetem of truncated hIL 6 was established which would be useful for preparation of IL 6 largely.

关 键 词：截短型人IL-6 原核表达 大肠杆菌 表达载体 构建 克隆载体 基因工程 

分 类 号：Q782[生物学—分子生物学] Q786