Construction of the shuttle plasmid vector for fusion protein gene and expression in Hepatocellular Carcinoma cells

To construct the shuttle plasmid vector for tk and EGFP fusion protein gene and detect its expression in transfected liver cancer cell lines HepG2 cells.Recombinant expression vector was constructed by techniques of gene recombination and screening,and identified by restriction digestion and sequencing analysis.Then the recombinant shuttle plasmid was transfected into HepG2 cell by techniques of lipidosome transfection and its expression was detected by fluoroscopy and RTPCR.Cell killing after GCV application was determined by MTT.Identification of pDC316-tkEGFP-CMV by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target gene which was inserted into the recombinant was correct and the expression of EGFP in transfected cell was observed.The MTT assess showed that the obvious cytotoxicity of GCV to HepG2 cells which was transfected the shuttle plasmid vector carrying the tk and EGFP fusion protein gene.The shuttle plasmid vector carrying the tk EGFP fusion protein gene has been constructed successfully and the individually expressing in transfected HepG2 cells of tk and EGFP was not effected.The shuttle plasmid vector can be used as report gene for monitoring the expressing of tk and as therapy gene for the suicide gene therapy of HCC.