title: Construction of recombinant eukaryotic expression plasmid containing hepatitis B virus genome with partial deletion of the core promoter.
abstract: OBJECTIVE: To construct an eukaryotic expression vector containing the full-length genome with partially deleted core promoter of hepatitis B virus (HBV). METHODS: A linearized genome containing the entire HBV 3.5 kb mRNA transcriptional units (P3.8 I plasmid) was used, which initiated from the upstream sequences of the basic core promoter. The objective eukaryotic expression vector was constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identifcation was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by cloning and sequencing analysis. RESULTS: The eukaryotic expression vectors containing HBV genomes with 20/21 bp deletion (position 1 748/1 747 to 1 767) in the core promoter or with precore stop mutation at nucleotide 1896 as well were constructed successfully as confirmed by sequence analysis with RFLP. CONCLUSION: The recombinant expression vector may lay the foundation for further studies into the biological significance of the above mentioned mutations in vitro.
authors.affiliation: Department of Infectious Disease, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Peng
authors.forename: Kang-Xian
authors.initials: KX
authors.lastname: Luo
authors.forename: Yu-Long
authors.initials: YL
authors.lastname: Lin
authors.forename: Jin-Lin
authors.initials: JL
authors.lastname: Hou
authors.forename: Ya-Bing
authors.initials: YB
authors.lastname: Guo
authors.forename: Zhan-Hui
authors.initials: ZH
authors.lastname: Wang