Objective: To construct a recombinant lentivirus vector containing fusion gene NT4-p53(N15)-Ant and transfer it into HepG2 cancer cells for gene therapy.
Methods: The gene of p53(N15)-Ant was obtained by T-vector method. After restriction enzyme digestion, the interest gene of p53(N15)-Ant was inserted in pBV220/NT4 vector and fusion gene of NT4-p53(N15)-Ant was subcloned into the plasmid of lentivirus and cotransferred into HEK-293 cells with helper plasmid. The recombinant lentivirus was produced by homologous recombination of the above mentioned two plasmids in HEK-293 cells and its titer was measured by plaque-forming. The expression of LV. NT4-p53-Ant in transfected HepG2 cells was finally confirmed by reverse transcription polymerase chain reaction (RT-PCR) procedure. The effect of LV. NT4-p53(N15)-Ant on HepG2 cells was measured by a colorimetric 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibition effect on HepG2 cells of LV. NT4-p53(N15)-Ant and its potential mechanism was detected by light microscopy, electron microscopy, MTT, LDH-release assay and annexin V-PI double staining.
Results: The gene of p53(N15)-Ant was confirmed by restriction enzyme digestion and DNA sequencing. High titer of the recombinant lentivirus was obtained by homologous recombination in HEK-293 cell lines (1 x 10(11) pfu/ml), and the expression of NT4-p53(N15)-Ant gene in HepG2 cells was confirmed by RT-PCR. The viability of HepG2 cells was decreased to 83.4%, 46.9% and 33.9%, at 24 h, 48 h and 72 h, respectively, after infection by LV. NT4-p53(N15)-Ant. Compared with the LV. EGFP control group, there were significant differences (P < 0.01). The LDH level in HepG2 cells infected by LV. NT4-p53(N15)-Ant at 48 h, 72 h and 96 h after infection was 682 IU/L, 815 IU/L and 979 IU/L, respectively, significantly increased than that in the LV. EGFP group (P < 0.01), indicating the cell membrane destruction.
Conclusion: The recombinant lentivirus vector encoding gene NT4-p53(N15)-Ant is successfully constructed in this experiment by molecular cloning and recombination in vitro techniques, and the results suggested that this fusion gene has an anti-tumor effect, which provides the basis for further research on recombinant adenovirus for cancer gene therapy.