Abstract: To construct the eukaryotic expression vector containing Herpes simplex virus thymidine kinase (HSV-TK) and interleukin2(IL-2) double genes and observe its expression in human pulmonary adenocarcinoma cell A549. Methods The sequences of multiple clone site (MCS) was synthesized and inserted to the vector pSNAV. HSV-TK, internal ribosome entry site (IRES), IL-2 fragments were respectively obtained from different plasmids, then they were inserted into the MCS using restriction enzyme to get the recombinant plasmid pSNAV-TK-IRES-IL2(pMTII). After identified by restriction enzyme digestion analysis and DNA sequencing, the recombinant plasmid was transfected into A549 (A549/pMTⅡ). The transcription of HSV-TK and IL-2 genes in the A549/pMTⅡ cells were detected by RT-PCR.The secretion of IL-2 in the A549/pMTⅡ cells was deteded by ELISA; the cytotoxicity of GCV (gancyclovir) on A549/pMTⅡ cells was observed by light microscope and MTT assay. Results Restriction enzyme digestion analysis, DNA sequencing, RT-PCR amplification and ELISA indicated that the recombinant plasmid pMTⅡ was constructed successfully and the target genes linked by IRES can express in cell A549. The A549/pMTⅡ cells administered ganciclovir (GCV) demonstrated obvious lethal effect by light microscope and MTT assay. Conclusion The eukaryotic expression vector pMTⅡ containing HSV-TK and IL-2 double genes was constructed successfully and provided a basis for further study of the double genes therapy for lung carcinoma.