en میکروب شناسی Microbiology <p style="text-align: justify;"><em><strong>Background:</strong></em> 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.</p> <p style="text-align: justify;"><em><strong>Methods:</strong></em> 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.</p> <p style="text-align: justify;"><em><strong>Results:</strong></em> 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.</p> <p style="text-align: justify;"><em><strong>Conclusion:</strong></em> 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.</p> Hepatitis Delta Virus, L-HDAg, SOEing-PCR 28 34 http://rbmb.net/browse.php?a_code=A-10-1-17&slc_lang=en&sid=1 Behnaz Forouhar Kalkhoran 10031947532846005955 10031947532846005955 No Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Farida Behzadian fbehzadian@yahoo.com 10031947532846005956 10031947532846005956 Yes Department of Molecular Genetics, Research Center for Sciences and Biotechnology, Malek Ashtar University, Tehran, Iran Farzaneh Sabahi 10031947532846005957 10031947532846005957 No Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Mohsen Karimi 10031947532846005958 10031947532846005958 No Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran Hesam Mirshahabi 10031947532846005959 10031947532846005959 No Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran