Construction of Eukaryotic Expression Vector with HIV-1 Integrase for Expression in Hela Cells / 昆明医科大学学报

ABSTRACT

Objective To construct recombinant eukaryotic expression vector for EGFP fused HIV-1 integrase expression. Methods Wild type of HIV-1 integrase gene was cloned into eukaryotic expression vector-pcDNA6/V5-HisA. After restricted enzyme mapping, PCR confirmation and sequence confirmation, the recombinant plasmid was transfected into HeLa cells with Lipofectamine2000. After 24 hours, the expression of integrase was examined by immunofluorescence with confocal fluorescent microscopy. The cells were fixed with 4%paraformaledhyde. The cell nuclei were stained with Propidium Iodide (PI). Then the expression was imaged and analyzed with Confocal Microscopy. Results The integrase expressed significantly in HeLa cells in 24 hours after transfection. Integrase was expressed and localized into nuclei mainly. After fixed with 4% paraformaldehyde, the cell nuclei were stained with PI. When nuclei were showed in red in normal cells, the nuclei with integrase over expression turned yellow or orange. Conclusion The construction of eukaryotic expression vector of integrase was successful. Integrase was expressed and localized into nuclei mainly after transfection in HeLa cells.