To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to effectively kill HeLa cells. Two tetracycline operator(TetO2) was cloned into pcDNA3.1 and a cassette was made for a cytomegalovirus-type 2 tetracycline operator(CMV-TetO2) promoter, and the obtained vector was named pcDNA3.1-CMV-TetO2. Herpes simplex virus thymidine kinase(HSV-tk) gene and tetracycline repressor(TR) gene were cloned into pcDNA3.1-CMV-TetO2 and the two genes were linked with internal ribosome entry site(IRES) to gain a vector named pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR. The HeLa cells were stablly transfected with pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR plasmid. The expression of HSV-tk and TR were detected by RT-PCR, the tumorcidal activity of HSV-tk/GCV was determined by MTT assay. In Hela cells transfected with the above plasmid vector, HSV-tk gene and TR gene can be expressed lowly and the concentration of GCV producing a 50% decrease in cell viability was about 50 μg/mL without adding deoxycycline; in contrast, the expessions of HSV-tk gene and TR gene increased significantly and the concentration of GCV producing a 50% decrease in cell viability was about 5 μg/mL with adding deoxycycline. Therefore tetracycline can regulate the expression and tumorcidal activity of HSV-tk gene in HeLa cells with this single plasmid vector. To construct a single tetracycline-regulatable plasmid vector based on the double tetracycline-regulatable plasmid vector system for regulating HSV-tk gene expression so as to kill HeLa cells. Two tetracycline operator (TetO2) was cloned into pcDNA3.1 and a cassette was made for a cytomegalovirus-type 2 tetracycline operator (CMV-TetO2) promoter, and the obtained vector was named pcDNA3.1-CMV-TetO2. Herpes simplex virus thymidine kinase (HSV-tk) gene and tetracycline repressor (TR) gene were cloned into pcDNA3.1-CMV-TetO2 and the two genes were linked with internal ribosome entry site (IRES) to gain a vector named pcDNA3.1-CMV-TetO2-HSV-tk- IRES-TR. The HeLa cells were stablly transfected with The expression of HSV-tk and TR were detected by RT-PCR, the tumorcidal activity of HSV-tk / GCV was determined by MTT assay. In Hela cells transfected with the above plasmid vector, HSV-tk gene and TR gene can expressed lowly and the concentratio n of GCV producing a 50% decrease in cell viability was about 50 μg / mL without adding deoxycycline; in contrast, the expessions of HSV-tk gene and TR gene increased significantly and the concentration of GCV producing a 50% decrease in cell viability was about 5 μg / mL with adding deoxycycline. Thus tetracycline can regulate the expression and tumorcidal activity of HSV-tk gene in HeLa cells with this single plasmid vector.