Cloning and implication of eukaryotic expression vectors containing hepatitis B virus genomes with partial deletion in the core promoter / 中华传染病杂志

ABSTRACT

Objective In order to further study the influence of a mutant on viral replication and transfection, a eukaryotic vector with mutation of 20/21 bp deletion (1748/ 1747 to nt 1767) in core promoter region and precore stop mutation (nt.1896) was constructed. Methods A linearized genome containing the entire HBV 3.5kb mRNA transcriptional units (P3.8Ⅰ vector) and initiating from the basic core promoter upstream sequences was used as a tool, the objective eukaryotic vectors were constructed by the molecular cloning and PCR based site directed mutagenesis in vitro. The capability of progeny virus production and transcription were examined with Southern blot and Northern blot analysis respectively, after transfection of the recombinant HBV plasmids into HepG2 cells by using liposome. Results The eukaryotic vectors were constructed successfully and their sequences were confirmed by clone sequencing. Both Southern and Northern blotting of DNA and RNA extracted from the transfected cells showed markedly reduced mutant activity to produce progeny virus, to transcript both 3.5kb precore/pregenome mRNA and 2.1kb preS/S mRNA. Conclusions The levels of replication and transcription are markedly reduced in the mutant compared with those in wild type HBV.