Objective:To isolate and clone the Negative factor(Nef) gene of human immunodeficiency virus 1(HIV-1),and to investigate its expression in the BCBL-1 cells and NIH/3T3 cells. Methods:A pair of PCR primers for Nef gene with EcoRⅠ and SalⅠ restriction enzyme cut sites was designed according to the sequence registered in GenBank. Then the genome of plasmid PCINL was taken as template and Nef gene was amplified using PCR. Subsequently,amplified gene fragments were digested with the two enzymes mentioned above,and then cloned into pCI-neo vector to create recombinant eukaryotic expression plasmid designated as pNefF,which was introduced with Flag sequence to facilitate the detection of protein. After identification with enzyme digestion and nucleotide sequences analysis,recombinant pNefF DNA was transient transfected into BCBL-1 cells and NIH/3T3 cells. The expression of pNefF mRNA and protein in BCBL-1 cells was detected by RT-PCR and Western blot,respectively. Results:Nucleotide sequences analysis indicated that the isolated and cloned pNefF sequence length was 651 bp. The isolated K9 sequence was 100% homology with Nef gene previously registered in GenBank. The specific bands were both detected at the expected place by RT-PCR and Western blot. Conclusion:Nef gene could be correctly expressed in BCBL-1 cells and NHT/3T3 cells.