关键词: 肝炎,乙型;病毒核心蛋白质类;遗传载体;基因表达

Abstract:

Objective  To construct two eukaryotic expression vectors containing the HBc gene and to observe their expressions in the human hepatoma cell line. Methods  We used pUC19／3-0HBV as a template to amplify the HBc gene with the PCR method. pcDNA3-0 and pEGFP-N1 were introduced to construct the expression vectors of pcDNA-HBc-HA and pEGFP-HBc, respectively. PCR, restriction endonuclease digestion and DNA sequencing were used to select the positive plasmids. Then, BEL7402 cells were separately transfected with pcDNA-HBc-HA, pEGFP-HBc and their empty control vectors by liposome. The HBc mRNA and protein levels were further determined by RT-PCR, Western blot and a fluorescent microscope. Results  The recombinant plasmids containing HBc gene were successfully constructed. Single and double enzyme digestion showed that both the recombinant plasmids contained the inserted gene fragments with the anticipated molecular weight. RT-PCR showed that high level of HBc mRNA was detected in BEL7402 cells transfected with pcDNA-HBc-HA and pEGFP-HBc expression vectors, while there was no HBc gene expression in control cells transfected with pcDNA3-0 or pEGFP-N1. The green fluorescence protein was expressed in the pEGFP-HBc group and the pEGFP-N1 group, and HBc-HA protein was detected in the pcDNA-HBc-HA group by Western blot. Conclusion  Two eukaryotic expression vectors with the HBc gene were successfully constructed and both of them were effectively expressed in BEL7402 cells.

Key words: Hepatitis B; Viral core proteins; Genetic vectors; Gene expression