title: Expression of NS3/NS4A Proteins of Hepatitis C Virus in Huh7 Cells Following Engineering Its Eukaryotic Expression Vector.
abstract: BACKGROUND
  Although the development of novel therapeutic regimens to combat hepatitis C virus (HCV) infection have been speeded up with successful results, no efficient vaccines exist yet.
  
  
  OBJECTIVE
  This study aimed to construct a eukaryotic expression vector encoding nonstructural proteins, NS3/NS4A, of HCV genotype 3a, and evaluate its expression on Huh7 cell surface.
  
  
  METHODS
  The NS3/NS4A sequence was isolated from a patient with HCV-3a chronic infection, cloned into intermediate vector pTZ57R/T, and then used for engineering a mammalian expression vector, pDisplay, to direct the respective protein to the secretory pathway and anchor it to the plasma membrane. The expression of the protein in Huh7 cell, which was transiently transfected with the vector using Lipofectamine, was determined by immunocytochemical staining assay with fluorescein isothiocyanate (FITC)-conjugated antibodies to the HA/myc tags located besides the fusion fragment.
  
  
  RESULTS
  The results showed that the fragment was successfully amplified and cloned into a eukaryotic expression vector. Sequencing and enzyme digestion analysis confirmed the cloned gene completion and its correct position in the pDisply-NS3/NS4A plasmid. Immunocytochemical staining revealed that the target protein was expressed as a membrane-anchored protein in the Huh7 cells.
  
  
  CONCLUSIONS
  This study can serve as a fundamental experiment for the construction of a NS3/NS4A eukaryotic expression vector and its expression in mammalian cells. Further research is underway to evaluate the fragment immunogenicity in lab animal models.
authors: Professor Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, IR Iran; Student Research Committee, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors: Mohammad Amin
authors: MA
authors: Behzadi
authors: Professor Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors: Abdolvahab
authors: A
authors: Alborzi
authors: Professor Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors: Gholamreza
authors: G
authors: Pouladfar
authors: Stem Cell and Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran; Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors: Mehdi
authors: M
authors: Dianatpour
authors: Professor Alborzi Clinical Microbiology Research Center, Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors: Mazyar
authors: M
authors: Ziyaeyan
keywords: Expression; Hepatitis C Virus; NS3; NS4A