RECK基因真核表达载体的构建及在HepG2细胞中的表达

Construction of RECK gene eukaryotic expression plasmid and its expression in human HepG2 cells

目的构建RECK(reversion-induc ing-cyste ine-rich prote in w ith Kazal motifs)基因的真核表达载体,通过脂质体介导法转染人肝癌细胞株HepG2并获得高表达RECK蛋白的细胞克隆。方法用RT-PCR方法扩增出人RECK基因,构建真核表达载体pcDNA3-RECK,采用脂质体介导法将重组质粒导入体外培养的HepG2细胞,RT-PCR和W estern b lot检测转染细胞和未转染细胞中RECK基因mRNA及蛋白质的表达。结果本实验成功构建了真核表达载体pcDNA3-RECK,并用脂质体介导的方法获得了高稳定表达RECK的细胞克隆;W estern b lot显示转染前的细胞未检测到RECK基因mRNA及蛋白质表达,但转染后表达量明显增高。结论重组质粒pcDNA3-RECK经转染能在HepG2细胞中高效表达,为进一步研究RECK对肝癌细胞的生物学影响奠定了基础。

Objective To construct eukaryotic expression plasmid of reversion-inducing-cysteine-rich protein with Kazal motifs （RECK） and establish httrnan HepG2 line monoclonal cells with the stable expression of RECK gene by transfection. Methods Human RECK gene was amplified by RT-PCR The recombi nant expression plasmid pcDNA3-RECK was constructed, and then introduced into HepG2 cell line by liposome method. Western blotting was applied to detect the RECK expression in transfected and non-transfected cells. Results The recombinant expression plasmid pcDNA3 RECK was constructed successfully. In HepG2 cell line transfected with the plasmid, RT-PCR and Western blotting confirmed that HepG2 cell lines didn＇t express RECK, but the level was much higher in transfected cells. Conclusion RECK can be expressed high effectively in HepG2 cell lines transfected with recombinant plasmid pcDNA3-RECK,which provides the basis for further study on the RECK function in HepG2.