Construction and Eukaryotic Expression of Recombinant Large Hepatitis Delta Antigen
سال انتشار: 1392
نوع سند: مقاله ژورنالی
زبان: انگلیسی
مشاهده: 177
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شناسه ملی سند علمی:
JR_RBMB-2-1_003
تاریخ نمایه سازی: 10 شهریور 1400
چکیده مقاله:
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 pcDNA۳.۱ and transfected to Huh۷ and HEK ۲۹۳ 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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نویسندگان
Behnaz Forouhar Kalkhoran
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Farida Behzadian
Department of Molecular Genetics, Research Center for Sciences and Biotechnology, Malek Ashtar University, Tehran, Iran
Farzaneh Sabahi
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Mohsen Karimi
Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran
Hesam Mirshahabi
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran