Construction of coexpression vector for HSV-1 thymidine kinase and green fluorescent protein

Objective To construct a functional TKGFP fusion protein therapeutic system for monitoring therapeutic transgene expression by noninvasive imaging of HSV-1-tk marker gene expression and establishing basis for the antitumour studies of Chinese medicine cooperating with suicide gene therapeutic system. Methods The eukaryotic HSV1-tk cDNA and EGFP fusion gene expression vector pTKGFP was constructed by using DNA recombinant technique. The recombinant plasmid was identified by restriction endonuclease digestion and DNA sequencing,and then mixed with PolyFect Transfection reagent and transfected into the melanoma cell line B16. A standard fluorescence microscope was used for fluorescent detection. MTT assay was used to determine the ganciclovir(GCV) sensitivity of transfected B16. Results The results of restriction endonuclease digestion and DNA sequencing showed that tk gene had been connected with GFP gene and there had been no shift-mutation in the linkage site. The levels of EGFP-expression were assessed days after transfection and were visually of similar intensity in TKGFP and native GFP transfected cells. The intracellular expression pattern of the TKGFP was different from the native EGFP. TKGFP was mostly concentrated in the nucleus,whereas the native EGFP displayed a predominantly cytoplasmic distribution. GCV sensibility examination showed that the pTKGFP transfected cells were much more sensitive to GCV than the pEGFP-N1 transfected cells in a dose-dependent manner. Conclusion A TKGFP fusion protein expression vector has been obtained successfully,which can be used for the antitumour studies of Chinese medicine cooperating with suicide gene therapeutic system.