title: Construction and eukaryotic expression of recombinant large hepatitis delta antigen.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Behnaz
authors.initials: B
authors.lastname: Forouhar Kalkhoran
authors.affiliation: Department of Molecular Genetics, Research Center for Sciences and Biotechnology, Malek Ashtar University, Tehran, Iran.
authors.forename: Farida
authors.initials: F
authors.lastname: Behzadian
authors.affiliation: Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Sabahi
authors.affiliation: Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Karimi
authors.affiliation: Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Hesam
authors.initials: H
authors.lastname: Mirshahabi
keywords: Hepatitis Delta Virus; L-HDAg; SOEing-PCR