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

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

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:   (5109 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]   (1947 Downloads)    
Subject: Microbiology
Received: 2013/05/22 | Accepted: 2013/07/10 | Published: 2013/10/30

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2015 All Rights Reserved | Reports of Biochemistry and Molecular Biology

Designed & Developed by : Yektaweb