Molecular Biology

, Volume 46, Issue 2, pp 226–235

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

Authors

    • Department of Biology, Science and Research BranchIslamic Azad University
  • F. Roohvandv
    • Department of Hepatitis and AIDSPasteur Institute of Iran
    • National Recombinant Gene Bank (NRGB)Pasteur Institute of Iran
  • M. R. Aghasadeghi
    • Department of Hepatitis and AIDSPasteur Institute of Iran
  • A. Eidi
    • Department of Biology, Science and Research BranchIslamic Azad University
  • S. Amini
    • Department of Hepatitis and AIDSPasteur Institute of Iran
  • F. Motevalli
    • Department of Hepatitis and AIDSPasteur Institute of Iran
    • National Recombinant Gene Bank (NRGB)Pasteur Institute of Iran
  • S. M. Sadat
    • Department of Hepatitis and AIDSPasteur Institute of Iran
  • A. Memarnejadian
    • Department of Hepatitis and AIDSPasteur Institute of Iran
  • G. Khalili
    • Department of ImmunologyPasteur Institute of Iran
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
Download to read the full article text

Copyright information

© Pleiades Publishing, Ltd. 2012