Construction of the vector for fusion protein gene driven by IGF-II P3 promoter and its expression in hepatocellular carcinoma cells

AIM: To construct the shuttle plasmid vector for thymidine kinase(tk) and EGFP fusion protein gene driven by IGF-Ⅱ P3 promoter,and investigate the specific killing effect of the HSV-tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro.METHODS: Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening,and identified by restriction digestion and sequencing analysis.Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT-PCR.Cell killing after ganciclovir(GCV) application was determined by MTT.RESULTS: Identification of pDC316-tkEGFP-P3 by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target genes which were inserted into the recombinant were correct.It was found that enhanced green fluorescence protein could only be seen in HepG2 cells,but not in HeLa cells.The results of RT-PCR showed that only two bands could be seen in the samples of pDC316-tkEGFP-P3 transfected HepG2 cells.The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells.CONCLUSION: The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.