Construction of recombinant plasmid of duck hepatitis B virus and expression

ObjectiveTo construct the recombinant plasmid containing 115-fold-overlength genome of DHBV and obtain the DHBV inocula collected from culture supernatant of DHBV transfected the avain LMH line.Methods414 kb fragment of DHBV genome,derived from pMD-DHBV and DHBV positive serum,was cloned into Sac I and Not I site of the vector PCR2.1 to construct the recombinant plasmid PRC2.1-DHBV1.5.And to identify its inserted direction by endonucleases digestion.The avain LMH hepatoma cells were transfected with plasmid PCR2.1-DHBV1.5 by using Lipofectine TM2000 transfection reagent.ResultsIt was successful in the recombinant plasmid PRC 2.1-DHBV1.5 containing 1.5-fold-overlength genome of DHBV.All expected DHBV replicative intermediates were verified by Southern blot analysis in the supernatant of L MH cells.And the DHBV could be observed by electron microscope.ConclusionThe recombinant plasmid PRC2.1-DHBV1.5 containing a competent-replication,which initiates viral replication efficiently in infected avian hepatoma cells,is expected as a useful source of DHBV inoculum in vivo.