Construction of eukaryotic plasmids of rat FasL full length cDNA and role of FasL transfection in induction of in vitro apoptosis of HepG2 cells

Objective To construct the eukaryotic expression vector and observe the effect of FasL gene transfection on induction of apoptosis of HepG2 cells.Methods RT-PCR and TA cloning technique were used to amplify the rFasL full length cDNA from rat testis cells,and then a eukaryotic expression vector pDC315 containing rFasL cDNA was constructed.Plasmid was transfected into HepG2 cells.The cells were harvested after 48h to identify the expression of plasmids by RT-PCR and Western blot.Meanwhile,two groups of HepG2 cells were respectively transfected with pDC315 plasmid as a plasmid control group and with H2O as a blank control group.After 48h of culture,the three groups of cells were counted,compared and analyzed.Results The sequence of cloned rFasL cDNA was consistent with that in GenBank.The HepG2 cells transfected with rFasL eukaryotic expression vector could express rFasL mRNA and protein.A large number of HepG2 cells died in the pDC315-rFasL experimental group.Conclusion The rFasL cDNA can be cloned and its eukaryotic expression vector can be constructed,indicating that rFasL gene can be expressed in HepG2 cells transfected with pDC315-rFasL.HepG2 cells expressing FasL induce apoptosis of neighbor-or auto-HepG2 cells,leading to their death.