Establishment of HIV-1 Nef stable expressed cell strain in THP-1 cells and the activity detection

To investigate the effects of HIV-1 negative regulate factor(Nef) gene on THP-1 cells,we established Nef stable expressing cells.Plasmids pcDNA 3.1(+)-Nef and pcDNA 3.1(+) were stably transfected into THP-1 cells,respectively.Then the expression and location of Nef protein were confirmed by RT-PCR,Western blotting and cell immunofluorescence.Fluorescent reporter gene was transfected into THP-1 Nef and THP-1 3.1 cells respectively by Co-transfection or evaluating the biological activity of Nef protein.Finally we obtained the stable Nef expressing THP-1 cell line through G418 selection.RT-PCR and Western blotting showed that the Nef successfully expressed in THP-1 cells,while cell immunofluorescence showed that the Nef protein was mainly located in the cytoplasm of THP-1-Nef cells.RLU values of THP-1-Nef and THP-1-3.1 cells,which transfected HIV-1 LTR-Luc and NFκB-Luc reporter gene,were detected by fluorescence microplate reader.THP-1 Nef stable expressed cells are estalished and identified successfully,which will lay the foundation for the further mechanisms studyiat the results indicate that Nef stble expressing.