Molecular Biology

, Volume 46, Issue 2, pp 226–235

Expression and characterization of Escherichia coli derived hepatitis C virus ARFP/F protein

  • F. Baghbani-arani
  • F. Roohvandv
  • M. R. Aghasadeghi
  • A. Eidi
  • S. Amini
  • F. Motevalli
  • S. M. Sadat
  • A. Memarnejadian
  • G. Khalili
Molecular Biology of the Cell

DOI: 10.1134/S0026893312020033

Cite this article as:
Baghbani-arani, F., Roohvandv, F., Aghasadeghi, M.R. et al. Mol Biol (2012) 46: 226. doi:10.1134/S0026893312020033

Abstract

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called “ARFP/F”, or “core+1,” protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11–161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the lb-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.

Keywords

hepatitis C virusARFP/FCore+1expressionimmunization

Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  • F. Baghbani-arani
    • 1
  • F. Roohvandv
    • 2
    • 3
  • M. R. Aghasadeghi
    • 2
  • A. Eidi
    • 1
  • S. Amini
    • 2
  • F. Motevalli
    • 2
    • 3
  • S. M. Sadat
    • 2
  • A. Memarnejadian
    • 2
  • G. Khalili
    • 4
  1. 1.Department of Biology, Science and Research BranchIslamic Azad UniversityTehranIran
  2. 2.Department of Hepatitis and AIDSPasteur Institute of IranTehranIran
  3. 3.National Recombinant Gene Bank (NRGB)Pasteur Institute of IranTehranIran
  4. 4.Department of ImmunologyPasteur Institute of IranTehranIran