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Vol 46(2012) N 2 p. 226-235;
F. Baghbani-arani1, F. Roohvand2,3*, M.R. Aghasadeghi2, A. Eidi1, S. Amini2, F. Motevalli2,3, S.M. Sadat3, A. Memarnejadian3, G. Khalili4

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

1Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, 16535446, Iran
2Department of Hepatitis & AIDS, Pasteur Institute of Iran, Tehran, 1316943551, Iran
3National Recombinant Gene Bank (NRGB) ,Pasteur Institute of Iran, Tehran, 1316943551, Iran
4Department of Immunology, Pasteur Institute of Iran, Tehran, 1316943551, Iran

*rfarzin@pasteur.ac.ir
Received - 2011-03-29; Accepted - 2011-07-05

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 1b-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.

Hepatitis C virus, ARFP/F, Core +1, expression, immunization



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