title: A simple and efficient strategy for the de novo construction of greater-than-genome-length hepatitis B virus replicons.
abstract: Hepatitis B virus (HBV) uses small covalently closed circular DNA for transcription and replication; linearization at any site would destruct at least one HBV open reading frame and interrupt the virus life cycle. Therefore, greater-than-genome-length (GGL) HBV replicons have been widely used for HBV replication studies. However, the existing strategies for the de novo construction of GGL HBV replicons are too complex to implement, especially when multiple replicons need to be cloned. In this study, the pHBV-basic plasmid was constructed for efficient cloning of GGL HBV replicons; changing the orientation of the site for type IIs restriction enzyme SapI in this plasmid reduced the de novo construction of various GGL HBV replicons to only one to three steps. Furthermore, Q5 high-fidelity DNA polymerase was found to be ideal for HBV genome amplification. In vitro experiments showed that the HBV replicon containing 1.31 genome copies replicated with better efficiency as evidenced by the titers of HBV DNA and HBsAg and HBeAg markers. The vector described in this study could serve as a powerful vehicle for in vitro and in vivo investigation of HBV replication.
authors.affiliation: Department of Biotechnology, Beijing Institute of Radiation Medicine, no. 27 Taiping Road, Haidian District, Beijing 100850, China. Electronic address: xjwang@bmi.ac.cn.
authors.forename: Xue-jun
authors.initials: XJ
authors.lastname: Wang
authors.affiliation: Department of Biotechnology, Beijing Institute of Radiation Medicine, no. 27 Taiping Road, Haidian District, Beijing 100850, China.
authors.forename: Xiu-juan
authors.initials: XJ
authors.lastname: Zhang
authors.affiliation: Department of Biotechnology, Beijing Institute of Radiation Medicine, no. 27 Taiping Road, Haidian District, Beijing 100850, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Hu
authors.affiliation: Department of Biotechnology, Beijing Institute of Radiation Medicine, no. 27 Taiping Road, Haidian District, Beijing 100850, China.
authors.forename: Ting-yu
authors.initials: TY
authors.lastname: Zhang
authors.affiliation: Department of Biotechnology, Beijing Institute of Radiation Medicine, no. 27 Taiping Road, Haidian District, Beijing 100850, China. Electronic address: sqwang@bmi.ac.cn.
authors.forename: Sheng-qi
authors.initials: SQ
authors.lastname: Wang
mesh_terms: D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D016679:Genome, Viral; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D010957:Plasmids; D012093:Replicon; D017422:Sequence Analysis, DNA; D014773:Virology / Q000379:methods*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens
keywords: Construction strategy; Greater-than-genome-length; Hepatitis B virus; Q5 high-fidelity DNA polymerase; Replicon