| [Objective] To construct the lentiviral RNA interference (RNAi) vector of human VEGF gene and realize the expression in human liver cancer cells MHCC97L.[Methods] According to the sequence of human VEGF gene, the complementary DNA chains which contain the target sequence were designed and synthesized, and then were connected to linear plenti6.3-MIR carriers in order to construct the miRNA lentiviral vector plasmid. The plasmid was transformed to DH5α cells, and the sequence were verified;293T cells were infected by the lentiviral vector plasmid with liposome mediated, lentiviral vectors were packaged, and the titer was measured. The human liver cancer cells MHCC97L were infected by the lentiviral vectors, and the VEGF expression in RNAi group (MHCC97-200), the negative control group(MHCC97-NEGA) and the blank control group (MHCC97-blank) were detected by Real-time PCR, to ensure the jamming effect of lentiviral vector plasmid to VEGF expression. Results The VEGF RNAi lentiviral vector plasmid is constructed successfully. The lentiviral vector was packed successfully, and the virus titer is3.23×109TU/ml.The VEGF gene expression is reduced by72.2%at mRNA level in MHCC97-200group.[Conclusion] The lentiviral RNAi vector of human VEGF gene is constructed successfully, and expressed effectively in MHCC97L cells. |
