人源性肝细胞生长因子稳定转染细胞株的建立(英文)

Building of stably transfected cell strain on human hepatocyte DNA synthetic stimulating factor

目的通过实验获得人源性肝细胞生长因子(hHDSSF)稳定转染细胞株,为重组产品的获得奠定基础。方法通过脂质体包裹重组质粒pcDNA3。1hisB-HDSSF转染Hela细胞,以G418筛选转染细胞;进而通过RT-PCR和NorthernBlot鉴定G418筛选后的单克隆抗性细胞株。结果重组质粒pcDNA3.1hisB-HDSSF转染Hela细胞后,第20天可见G418单克隆抗性细胞株的形成。G418抗性Hela细胞株RT-PCR扩增出600bp左右的目的条带;NorthernBlot获得了明显的阳性杂交影。结论我们的实验证实HDSSF表达重组体被成功转入Hela细胞,并稳定表达;从而获得了HDSSF稳定表达细胞株,为进一步研究HDSSF表达、蛋白纯化和测定其生物学活性奠定了基础。

Objective: To obtain human hepatocyte DNA synthetic stimulating factor (hHDSSF) stable transfected cell strain. Methods: Recombinant plasmid pcDNA3.1hisB-HDSSF transfected Hela cell by lipofectin, G418 was used for screening. The monoclonal resisting cell strain screened by G418 was confirmed with RT-PCR and Northern Blot. Result: The monoclonal cell strains with resistance to G418 were formed on 20th day after Hela cell was tansfected with pcDNA3.1hisB-HDSSF. A 600bp special fragment was amplified by RT-PCR in the monoclonal cell strains, and Northern Blot also showed obvious positive hybridization shadow in the cell strain. Conclusions: HDSSF expressive recombinant has been transfected successfully into Hela cells and expressed steadily, which sets up a base for further study of HDSSF expression, purification and its biological activity.