Construction Of Coexpression Vectors Containing HSV-tk, IL-2, TNF-αGenes And In Vitro Study On Kjlling Effects Of HSV-tk/GCV On Hep-2 Human Laryngocarcinoma Cells

Objective: The aim of this study was to construct coexpression vectors containing HSV-tk, IL-2 and TNF- a genes. We evaluated the killing effects of HSV-tk/GCV on Hep-2 human laryngocarcinoma cells in vitro. Methods: (1) The three genes were amplified respectively by PCR and the purified PCR products were ligated into the sequencing vector pGEM-T-Easy. The white clones were selected and the recombinant plasmid was purified, which was further identified by restriction endonucleases and was sequenced . By the method of directional cloning and introduction of IRES, the sequenced genes were subcloned into corresponding restriction sites of pLXSN in turn to generate different coexpression vectors. (2) HSV-tk gene was subcloned into retroviral vector pLXSN by recombinant DNA technology. pL(tk)SN was packaged with PA317 and selected in G418 to obtain the positive clones, which was able to produce stable retrovirus. Hep-2 was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed Hep/tk . The integration and expression of tk gene inHep/tk cells were identified by PCR and RT-PCR.The GCV killing effects on gene-modified cells were assessed by observing growth state of Hep/tk, MTT and FCM. Results: (1) By sequencing , restriction digestion and PCR we confirmed that the sequence, length, position and orientation of inserted genes were all correct. Then target vectors, pL(TI)SN and pL(TT)SN, were constructed successfully. (2) The retroviral vector pL(tk)SN was generated and stable titer producing cell CK, was established(1.6x106/ml). PCR and RT-PCR demonstrated that tk gene was integrated into the Hep/tk genome and expressed at the mRNA level. There was no significant difference in cell proliferation among Hep/tk, Hep/0 and Hep-2 cells. Hep/tk cells were highly sensitive to GCV ^specially in low concentration of GCV. Conclusions: We successfully constructed coexpression vectors containing HSV-tk, IL-2 and TNF- a genes .In vitro study showed that laryngocarcinoma cells were sensitive to HSV-tk/GCV system . These results suggest that HSV-tk/GCV system is an effective gene therapy and will lay some foundation for clinical treatment of laryngocarcinoma.