OBJECTIVE To construct a shuttle plasmid vector of fused herpes simplex virus thymidine kinase (HSV-tk) gene and enhanced green fluorescent protein (EGFP) gene driven by human insulin-like growth factor II (IGF-II) P3 promoter, and investigate the special killing effect of the HSV-tk/ganciclovir (GCV) system on hepatocellular carcinoma (HCC) cells. METHODS An adenovirus shuttle plasmid, pDC316-tkEGFP-CMV containing fused genes tkEGFP and an adenovirus shuttle plasmid pDC316-tkEGFP-P3 driven by IGF-II P3 promoter were constructed by techniques of gene recombination and screening, and identified by restriction digestion and sequencing analysis. Human hepatocellular carcinoma cells HepG2 and human cervical carcinoma cells HeLa were cultured and transfected with these 2 recombinant shuttle plasmids. RT-PCR was used to detect the mRNA expression of EGFP and HSV/tk. GCV of the final concentrations of 0, 1, 10, and 100 microg/ml respectively was added into the culture fluid of the HepG2 cells transfected with pDC316-tkEGFP-CMV or pDC316-tkEGFP-P3, and MTT method was used to detect the cell inhibition rate. RESULTS Digestion and sequencing analysis showed that the recombinant plasmid pDC316-tkEGFP-P3 accorded with the design. Fluorescent microscopy showed that EGFP was expressed only in the HepG2 cells, but not in the HeLa cells. RT-PCR showed that mRNA expression of EGFP and HSV/tk could be seen in both HepG2 and HeLa cells transfected with pDC316-tkEGFP-CMV or pDC316-tkEGFP-P3, however, only in the pDC316-tkEGFP-P3 transfected HepG2 cells, but not in the HeLa cells transfected with pDC316-tkEGFP-P3. MTT assay showed that GCV dose-dependently inhibited the 2 cancer cells, the inhibition rates of GCV of the final concentrations of 1, 10, and 100 microg/ml were 24.1% +/- 1.9%, 45.1% +/- 1.7%, and 69.4% +/- 3.6% in the HepG2 cells, and 25.1% +/- 1.6%, 49.3% +/- 1.1%, and 72.2% +/- 2.9% in the HeLa cells. However, the inhibition rates of the pDC316-tkEGFP-P3-transfected HepG2 cells by GCV of the final concentrations of 1, 10, and 100 microg/ml wee 19.8% +/- 1.3%, 36.2% +/- 2.0% and 48.7% +/- 1.9% respectively, all significantly lower than those of the pDC316-tkEGFP-CMV-transfected HepG2 cells (all P < 0.01), and no significant cell inhibition was found in the HeLa cells transfected with pDC316-tkEGFP-CMV. CONCLUSIONS A shuttle plasmid vector containing the tkEGFP fusion protein gene driven by IGF-II P3 promoter has been constructed successfully and its specific expression in HepG2 cells provides a sound basis for targeted gene therapy for HCC.