title: High-level production of a functional recombinant hepatitis B virus polymerase in insect cells with a baculovirus expression system.
abstract: HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerase gene to express the target protein. HBV polymerase expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.
authors.affiliation: Institute of Liver Diseases, Tongji Hospital, Tongii Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Wang
authors.forename: Linlin
authors.initials: L
authors.lastname: Gao
authors.forename: Fei
authors.initials: F
authors.lastname: Deng
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Jusheng
authors.initials: J
authors.lastname: Lin
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase