人胰岛素样生长因子-1的真核表达及其生物学功能研究(英文)

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 bolt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。

Objective: To construct IGF-1 expression vector and express it in CHO cells. Methods: Total RNA from human liver were prepared, and IGF gene by reverse transcriptase -polymerase chain reaction (RT-PCR) was amplified, then IGF-1 fragment to expression vector pSec Tag/FRT/V5-His was cloned, till the positive clone was verified by sequencing. Finally the positive plasmid to CHO cells was transfected by Lipofectamine, and medium for SDS/PAGE and Western Blot analysis was collected. Results: A 210-bp IGF-1 gene was successfully amplified, and a positive clone checked by sequence was got; Western Blot result suggested IGF-1 can be expressed in CHO cells. Conclusions: Type-1 insulin like growth factor was successfuly constructed and expressed. The recombinant IGF-1 can promote BMSC proliferation.