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

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.

Discovery of Protease Inhibitors of HIV-1 through Structure Based Virtual Screening / 昆明医科大学学报

Objective Through new virtual screening tools of PyRx to run AutoDock Vina to virtually screen the 20000 compounds in ZINC database,so as to discover new HIV protease inhibitors, and make a tentative study of the combination model of them with HIV protease. Methods The study focused on the targets of HIV protease, the virtual screening program of AutoDock Vina was used to virtually screen the compounds in ZINC database. It was differing from previous studies by using new virtual screening tools of PyRx to run AutoDock Vina. The HIV protease crystal structure (PDB ID:4phv) was downloaded from PDB and dealed with AutoDock Tools. Compound structure was downloaded from ZINC database and imported with PyRx, processed into format of pdbqt. The post-screen compounds were imported into AutoDockTools, and the data were outputted with PyMOL.Results There were 1000 drugs of small molecular compound for high-throughput screening from about 20000 compounds in the library. After screening for 3 times we found five highly active HIV protease inhibitors from the 1000 small molecular compounds.Conclusion The further development of the five new HIV protease inhibitors will contribute to the treatment and basic research of HIV,and provide new reference for structure-based virtual screening and discovery of HIV drugs.