To　produce　the　reverse　transcriptase　of　moloney　murine　leukemia　virus　(MMLV-RT)　through　gene　recombination,　MMLV-rt　gene　was　amplified　by　polymerase　chain　reaction　(PCR)　with　specifically　designed　primers　bearing　restriction　enzyme　sites.　Five　mutation　sites　increasing　the　solution　of　the　target　protein　were　introduced　through　Site-directed　mutation.　After　verification　by　sequencing,　the　gene　was　cloned　into　the　expression　vector　pET15b　to　construct　the　recombinant　plasmid　pET15b-MMLV-rt.　Purified　MMLV-RT　was　obtained　by　affinity　chromatography　(Ni3+-NTA　beads).　Molecular　weight　and　purity　of　MMLV-RT　were　analyzed　with　SDS-PAGE.　Enzyme　activity　was　characterized　with　RT-PCR.　We　successfully　constructed　the　recombinant　plasmid　pET15b-MMLV-rt　and　obtained　the　MMLV-RT　fusion　protein　with　6His　on　the　N-terminus.　Recombinant　protein　was　purified　through　Ni3+-NTA　beads　based　affinity　chromatography,　the　purity　of　which　was　96%.　The　Activity　of　the　enzyme　was　high.　MMLV-RT　of　96%　purity　was　obtained　with　the　prokaryotic　expression　technique,　which　serves　as　the　basis　for　mass　production　of　this　enzyme.　©　2008　Institute　of　Microbiology,　CAS　&　CSM,　All　rights　reserved.