类胰岛素生长因子-Ⅰ的克隆、表达、分离纯化与活性检测  

The Cloning, Expression, Purification and Bioactivity Identification of Recombinant IGF-Ⅰ

作　　者：魏辉俊[1] 黄秉仁[1] 蔡良琬[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所,北京100005

出　　处：《中国医学科学院学报》1997年第5期342-345,共4页Acta Academiae Medicinae Sinicae

摘　　要：把类胰岛素生长因子-Ⅰ（IGF－Ⅰ）cDNA重组于酵母游离体型表达载体YEpHC8，转化酵母后获得了IGF－Ⅰ的表达，并经Bio－Rex70和Bio－GelP10分离纯化后进行了重组蛋白的活性检测。The fragment containing IGF-I cDNA and cycl terminator from PSK-IGF-I plasmid wascloned into PSK43 SB treated by Hind N, Klenow and C1a I, generating a recombinant PSB-IGF-I (DH), which have double Hind N sites. This plasmid was treated by Hind N andKlenow, ligated to give a recombinant plasmid PSB-IGF-I(NH), which has no Hind N site.After analysed by restriction endonucleases and sequencing, the fusion site was verified to becorrect. This plasmid was excised and a BamH I -Cla I fragment containing yeast Q-factorpromotor, leader sequence, IGF-I cDNA and cycl terminator was cloned into yeast episomalplasmid vector YEpHC8, generating a recombinant plasmid YEpHC8-IGF-I. Transformedthis plasmid into yeast competence cells BJl99O by using LiAc method. The expressed prod-ucts were secrected into the medium broth and having the correct. molecular weight of 8 100on SDS-PAGE. The products of IGF-I were raised by using high-cell-density fermentation ofSaccharomyces cerevisiae. The cleared supernatant of yeast medium was applied to Bio-Rex7O resin, eluted with 1 mol/L ammonium acetate, pH8. O. Main peak was pooled and appliedto Bio-Gel P1O (200~400 mesh). The second peak was collected and get relatively pure IGF-I proteins. The biological activities of IGF I product was assayed in NIH3T3 cells by usingMTT method. The results show that the expressed IGF-1 can obviously stimulate NIH3T3cells to proliferate at the concentration ranging from 10ng/ml to 50ng/ml, suggesting thatthe protein has its biological activities.

关 键 词：基因表达 IGF-I 类胰岛素 生长因子 克隆 活性 

分 类 号：Q784[生物学—分子生物学]