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Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle
Published on: February 1, 2017
Construction and eukaryotic expression of recombinant large hepatitis delta antigen
Behnaz Forouhar Kalkhoran1, Farida Behzadian2, Farzaneh Sabahi1
1Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
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.
