Objective:To isolate and clone the vesicular stomatitis virus envelope glycoprotein(VSV-GP) gene and produce the VSV-GP pseudotyped Human Immunodeficiency Virus-1(HIV-1),investigating its ability to infect the BCBL-1 cells. Methods:A pair of PCR primers for VSV-GP gene with HindⅢ and BamHⅠ restriction enzyme sites was designed according to the sequence in GenBank. Then the VSV-GP gene was amplified from the plasmid pHCMV-G using PCR. Subsequently,amplified gene fragments were digested and cloned into pcDNA3.1(+) vector to create recombinant eukaryotic expression plasmid pVSV-GP. BCBL-1 cells were infected by VSV-GP pseudotyped HIV-1,which was harvested from HEK 293T cells cotransfected by pVSV-GP and pNL4-3.Luc.R-E-.Then BCBL-1 cells were lysed to calculate the relative luciferase unit(RLU), and the concentration of HIV-1 p24 antigen in the supernatant was determined by using a HIV-1 p24 antigen ELISA kit. Meanwhile,HEK 293T cells were lysed to detect the expression of HIV-1 p24 antigen by Western blott. Results:The isolated and cloned pVSV-GP sequence was 1 536 bp and was confirmed by sequencing. HIV-1 p24 antigen could be detected in the supernatant of VSV-GP pseudotyped HIV-1 infected BCBL-1 cells. The RLU of BCBL-1 cells infected by VSV-GP pseudotyped HIV-1 increased significantly compared with the negative control(P < 0.05), and the band of HIV-1 p24 antigen was detected by Western blott. Conclusion:VSV-GP pseudotyped HIV-1 was successfully produced and had the ability of infecting BCBL-1 cells.