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Molecular Cloning of Survivin Gene Promoter and Detecting Its Specific Activity in HeLa Cell
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Abstract:
Objective : To construct pGL3Basic eukaryotic expression vector containing survivin promoter gene and explore the activity of this survivin promoter in Hela cells. Methods: The survivin gene promoter was amplified by polymerase chain reaction and cloned into pGL3Basic vector to construct pGL3Basic eukaryotic expression vector containing survivin gene promoter (pGL3Basic/Surp). The purified pGL3Basic/Surp was transiently transfected into HeLa cell and vessel endothelial cell line EVC304 using liposome transfection reagent and the activity of survivin gene promoter was determined by adding luciferase substrate into transfected cells 48 h later. Results: About 1 kb gene fragment was amplified by PCR method from Hela cell genomic DNA and pGL3Basic/Surp vector was constructed successfully. The activity of luciferase reporter gene was 2074.2±78.5 in Hela and 9.7±1.1 in EVC304 48 h after transfection of pGL3Basic/surp vector. Conclusion: The high specific activity of constructed survivin promoter eukaryotic expression vector might be a potential therapeutic reagent for the treatment of malignant tumor.
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