Construction of recombinant replication competent plasmids containing HBV genome with mutations of precore and basic core promoter / 中华实验和临床病毒学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To construct a series of recombinant replication competent plasmids of hepatitis B virus (HBV) full-length genome with varying mutations of precore and basic core promoter.</p><p><b>METHODS</b>The plasmid containing the entire HBV (1.2 copies) genome was used to construct objective plasmids. The objective competent vectors were constructed by molecular cloning and PCR-based site-directed mutagenesis in vitro and confirmed with sequence analysis. After introducing the plasmids into hepatocellular carcinoma cell line (Huh7) by calcium phosphate transfection, the culture supernatant was collected to detect HBsAg and HBV DNA to analyze viral replication and expression.</p><p><b>RESULTS</b>Ten competent vectors with varying precore and basic core promoter mutations were constructed. After transfecting into Huh7, the vectors replicated and secreted viral particles.</p><p><b>CONCLUSIONS</b>Ten full-length competent HBV recombinants were obtained, which were harbouring varying mutations within precore and basic core promoter.</p>