人胰岛素样生长因子-1的真核表达及其生物学功能研究

Expression and functional analysis of type-1 insulin-like growth factor

目的:构建人胰岛素样生长因子1(IGF-1)分泌型真核表达载体,并检测表达产物的生物活性。方法:用RT-PCR法从人肝细胞克隆IGF-1基因,并将其连入Psec Tag/FRT/V5-His载体,用脂质体法转染CHO细胞,将转染细胞上清培养骨髓基质干细胞,用MTT法测定骨髓基质干细胞的增殖情况。结果:成功扩增210bp的IGF-1基因,表达产物经SDS-PAGE分析及Western blotting检测具有7.7kD特异性条带。骨髓基质干细胞的增殖随转染细胞上清的浓度、转染细胞上清培养时间的增加而增加。结论:成功构建分泌型IGF-1真核表达载体,其表达产物对骨髓基质干细胞具有促增殖作用。

AIM： To construct recombinant human IGF - 1 eukaryofic expression vector, and to study the hiological effect of rlGF - 1 by transfecting the vector into CHO cells. METHODS： Total RNA was prepared from human liver, and IGF - 1 gene was amplified by reverse transcriptase - polymerase chain reaction （ RT - PCR）. IGF - 1 fragment was inserted into the expression vector pSec Tag/FRT/V5 - His and the positive clone was verified by sequencing. Finally, the positive plasmid was transfected to CHO cells by Lipofectamine. The culture medium was analyzed by SDS/PAGE and Western blotting. The effect of rlGF - 1 on MSC proliferation was studied with the MTr method. RESULTS： The IGF - 1 expression vector was constructed and transfected into CHO cells. Western blotting results demonstrated that rlGF - 1 was expressed in CHO cells. The recombinant IGF - 1 promoted MSCS proliferation. CONCLUSION： We successfully constmcted the IGF - 1 expression vector and expressed type - 1 insulin - like growth factor with its biological function.