Gene　therapy　offers　the　promise　of　curing　the　HIV-infected　patients.　Specific,　potent,　and　sustained　short　hairpin　RNA　(shRNA)-mediated　gene　silencing　is　crucial　for　the　successful　application　of　RNA　interference　technology　to　therapeutic　interventions.　To　reduce　the　probability　of　viral　escape　mutants,　in　this　study,　we　constructed　lentiviral　vector　containing　vpr　and　tat　shRNA,　respectively,　furthermore　the　bispecific　lentiviral　vector　harboring　vpr　and　tat　shRNA　expression　cassettes　from　U6　promotor　and　H1　promotor　was　cotransfected　with　recombinant　plasmid　expressing　the　vpr　and　tat　gene.　The　result　showed　that　the　bispecific　lentiviral　vector　plvx-vpr-tatshRNA　could　inhibit　the　vpr　and　tat　effectively,with　ratios　of　89.20%　and　62.00%　respectively.　When　cotransfected　with　pNL4-3　in　293T　cell,　plvx-vpr-tatshRNA　showed　higher　efficacy　in　down　regulating　the　HIV　NL4-3　packaging　production　than　the　plvx-vprshRNA　or　plvx-tatshRNA　individually.　MT4　cell　clones　transduced　with　recombinant　lentiviral　vectors　were　screened　and　challenged　with　HIV　NL4-3.　P24　ELISA　test　showed　that　MT4　transduced　with　the　combinational　lentiviral　vector　could　inhibit　virus　replication　efficiently.