TY - JOUR JF - rbmb JO - rbmb.net VL - 2 IS - 1 PY - 2013 Y1 - 2013/10/01 TI - Construction and Eukaryotic Expression of Recombinant Large Hepatitis Delta Antigen TT - N2 - 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. SP - 28 EP - 34 AU - Forouhar Kalkhoran, Behnaz AU - Behzadian, Farida AU - Sabahi, Farzaneh AU - Karimi, Mohsen AU - Mirshahabi, Hesam AD - Department of Molecular Genetics, Research Center for Sciences and Biotechnology, Malek Ashtar University, Tehran, Iran KW - Hepatitis Delta Virus KW - L-HDAg KW - SOEing-PCR UR - http://rbmb.net/article-1-38-en.html ER -