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.

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@article{Kalkhoran2013ConstructionAE, title={Construction and eukaryotic expression of recombinant large hepatitis delta antigen.}, author={Behnaz Forouhar Kalkhoran and Farida Behzadian and F. Sabahi and Mohsen Karimi and Hesam Mirshahabi}, journal={Reports of biochemistry & molecular biology}, year={2013}, volume={2 1}, pages={28-34} }