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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 stable cell lines transfected with human hepatocyte growth factor (hHDSSF) through experiments and lay the foundation for obtaining recombinant products. Methods Hela cells were transfected with recombinant plasmid pcDNA3.1hisB-HDSSF by lipofectamine. The transfected cells were screened by G418, and the monoclonal cell lines were screened by RT-PCR and NorthernBlot. Results The HeLa cells transfected with recombinant plasmid pcDNA3.1hisB-HDSSF showed the formation of G418 monoclonal resistant cell line on the 20th day. The target band of about 600bp was amplified by RT-PCR in G418-resistant Hela cell line. Northern Blot showed obvious positive hybridization. Conclusion Our experiment confirmed that HDSSF expression recombinant was successfully transfected into Hela cells and stably expressed. Thus HDSSF stable cell line was obtained, which laid the foundation for the further study of HDSSF expression, protein purification and determination of its biological activity.