construction and eukaryotic expression of recombinant large hepatitis delta antigen

background: hepatitis delta virus (hdv) is a subviral human pathogen that exploits host rna editing activity to produce two essential forms of the sole viral protein, hepatitis delta antigen (hdag). editing at the amber/w site of hdv antigenomic rna leads to the production of the large form (l-hdag), which is required for rna packaging. methods: in this study, pcr-based site-directed mutagenesis by the overlap extension method was used to create the point mutation converting the small-hdag (s-hdag) stop codon to a tryptophan codon through three stages. results: sequencing confirmed the desirable mutation and integrity of the l-hdag open reading frame. the amplicon was ligated into pcdna3.1 and transfected to huh7 and hek 293 cell lines. western blot analysis using enhanced chemiluminescence confirmed l-hdag expression. the recombinant l-hdag localized within the nuclei of cells as determined by immunofluorescence and confocal microscopy. conclusion: because l-hdag requires extensive post-translational modifications, the recombinant protein expressed in a mammalian system might be fully functional and applicable as a tool in hdv molecular studies, as well as in future vaccine research.