Molecular Cloning Of Survivin Gene Promoter And Detecting Its Function In Tumor Special Gene Therapy

Objective: Molecular cloning of survivin gene promoter and construct siRNA eukaryotic expression vector under survivin promotor. Methods: (1)Approximately 1 kb of the survivin gene promoter region was amplified by polymerase chain reaction and cloned into pGEM-T Easy vector to generate the plasmid pGEM-T/surp which was confirmed by DNA sequencing. (2) The pGEM-T/surp and pGL-3 Basic vector were digested by SacI and HindIII enzyme and right size bands were gel received respectively, then survivin promoter gene fragment from pGEM-T/surp was relegated with linearized pGL-3 Basic vector to construct pGL-3 Basic eukaryotic expression vector bearing surviving gene promoter(pGL3-Basic/surp), which was further confirmed by double enzyme digestion. (3)The purified pGL3-Basic/surp was transiently transfected into HeLa cell and vessel endothelial cell EVC304 using liposome transfection reagent and the activity of survivin gene promoter was determined by adding luciferase substrate into transfected cells 48 hours later. (4)Two target siRNA gene segments were synthesized and cloned into pSilencer4.1-CMV neo vector respectively to construct two recombinant eukaryotic expression vectors: pSilencer4.1-CMV neo-S1 and...