Research Of Retroviral Vector Expressing Wtp53 Gene Driven By Human Telomerase Reserve Transcriptase Gene Promoter

Objective: In order to develop retrovirus targeting gene therapy against tumor, A retroviral vector expressing wtp53 gene driven by hTERT promoter was constructed.Methods: 1. We transformed JM109 with the plasmids pCMV-Neo — BamH-wtp53, pLHCX, phTERT-luc. They were extracted from the transformed bacteria, cut by restriction endonuclease and then demonstrated using agarose gel electrophoresis. 2. Construction of the plasmids: The plasmid phTERT-luc containing 319bp hTERT promoter gene fragment was digested then directedly ligated into pEGFP-N₂ with T₄DNA Ligase to construct the plasmid phTERT-EGFP-N₂; then the plasmid phTERT-EGFP-N₂ was cut with restriction endonuclease to get hTERT promoter and EGFP gene fragment directedly ligated into pLHCX that removed CMV IE promoter to construct pLHCX-hTERT-EGFP. The plasmid pCMV-Neo — BamH-wtp53 containing 1.8kb wtp53 gene fragment was digested with restriction endonuclease, then directedly ligated into pEGFP-C₁ with T₄DNA Ligase to construct the plasmid pEGFP-wtp53, chose the norientation ones; The plasmid phTERT-luc containing 319bp hTERT promoter gene fragment was digested , then directedly ligated into pEGFP-wtp53 to construct the plasmid phTERT-EGFP-wtp53; hTERT promoter and wtp53 gene fragment was digested with restriction endonuclease and directedly ligated into pLHCX that removel CMV IE promoter to construct pLHCX-hTERT-wtp53. 3. The specificity of plasmid pLHCX-hTERT-EGFP in tumor cell: According to the transfection procedure, Human colon cancer cell line SW620(hTERT promoter positive) and hTERT promoter negative...