Construction Of Lentiviral Vector Carrying TRAIL Gene And Its Expression In Hepatocarcinoma Cells

Objective:To construct lentiviral expression vector carrying TRAIL gene and realize its stable high expression in hepatocellular carcinoma cell line HepG2. To detect the proliferation of HepG2and observe the growth of TRAIL highly expressed in HepG2in mice.Methods:1. PCR and Double digestion were carried out to construct recombinant lentiviral vector pCDH-CMV-TRAIL-EF1-GFP-T2A-Puro;2. The293T cells were contransfected with the recombinant lentiviral vector and lentivirus package plasmid to produce lentiviral particles by lipofectamine method. The lentivirus was purified and virus titer was measured;3. Western blotting was used to detect the expressions of TRAIL protein;4. MTT assay was used to determine cells proliferation;5. To detect the growth of HepG2cells by nude mice experiment in vivo.Results:The recombinant lentiviral vector carrying TRAIL was confirmed by restriction end nuclease analysis and DNA sequencing. Virus titer reached to1.02×104ifμ/μL, and found that that the recombinant lentiviral transfected efficient in vitro when MOI=8. After puromycin selection and monoclony, the stable high expressions of TRAIL protein were confirmed by Western blotting. To detected the cell proliferation activity by MTT assay, we found that HepG2with stable high expressions of TRAIL protein was significantly lower than in HepG2and HepG2-con group (P<0.05). The tumor volume of HepG2-TRAIL-1was significant differences compared with other groups by nude mice experiment in vivo (P<0.01).Conclusion:Lentiviral vector carrying TRAIL gene has been successfully constructed and realizes its stable high expression in HepG2cells. It has proved that TRAIL gene can inhibit tumor cell growth by liver cancer cells and nude mice experiments.