Objective: To construct the recombinant lentivirus containing HIV-1 Nef gene and detect the protein expression in its target cell lines, such as 293T, BCBL-1 and EA.hy926, and explore the effect of Nef on cell proliferation. Methods:The fragment of Nef gene from expression vector pCI-neo-Nef was cloned into the lentivirus vector pHAGE-CMV-MCS-Izs-Green, then the recombinant vector pHAGE-Nef, packaging vector psPAX2 and envelope vector pMD2.G were cotransfected into the 293T cells. Culture media were harvested and filtered through a 0.45 μm filter to remove the cells. The viral titer was checked by observing the expression of green fluorescent protein (GFP). After infection of the recombinant lentivirus, the protein expression of Nef in 293T, BCBL-1 and EA.hy926 cells were detected by Western blot. The effect of Nef on cell proliferation was investigated by Cell Counting Kit-8 assay. Results:The recombinant lentivirus vector carrying HIV-1 Nef was constructed successfully. The viral titer was 1×107 TU/ml. 293T, BCBL-1 and EA.hy926 cells could be efficiently infected by it. The expression of Nef in these cell lines could be detected by Western blot. In addition, Nef protein could inhibit the proliferation of BCBL-1 and EA.hy926 cells. Conclusion:Recombinant lentivirus with high titer and efficient infection of 293T, BCBL-1 and EA.hy926 cells, could be obtained quickly and simply by using the lentivirus vectors system, and Nef expression could be detected in 293T, BCBL-1 and EA.hy926 cells infected by lentivirus-Nef. Furthermore, our study suggested that Nef protein could inhibit the proliferation of BCBL-1 and EA.hy926 cells.