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

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.