Volume 2, Number 1 (Vol.2 No.1 Oct 2013) | rbmb.net 2013, 2(1): 28-34 | Back to browse issues page



PMID: 26989717
PMCID: PMC4757064

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Forouhar Kalkhoran B, Behzadian F, Sabahi F, Karimi M, Mirshahabi H. Construction and Eukaryotic Expression of Recombinant Large Hepatitis Delta Antigen. rbmb.net. 2013; 2 (1) :28-34
URL: http://rbmb.net/article-1-38-en.html

Department of Molecular Genetics, Research Center for Sciences and Biotechnology, Malek Ashtar University, Tehran, Iran
Abstract:   (790 Views)

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.

Full-Text [PDF 320 kb]   (135 Downloads)    
Subject: Microbiology
Received: 2016/08/21 | Accepted: 2016/08/21 | Published: 2016/08/21

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