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Title of article :
Construction and eukaryotic expression of recombinant large hepatitis delta antigen
Author/Authors :
Forouhar Kalkhoran، Behnaz نويسنده Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran , , Behzadian، Farida نويسنده , , Sabahi، Farzaneh نويسنده , , Karimi، Mohsen نويسنده , , Mirshahabi، Hesam نويسنده Department of Virology, School of Medicinet ,
Issue Information :
دوفصلنامه با شماره پیاپی 0 سال 2013
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Abstract :
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.
Journal title :
Reports of Biochemistry and Molecular Biology (RBMB)
Journal title :
Reports of Biochemistry and Molecular Biology (RBMB)
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