A confocal-microscopic study on HCV core+1 protein expression
Subject Areas : Molecular MicrobiologyHassan Noorbazargan 1 , Atieh Hashemi 2 , Mohamadreza Aghasadeghi 3 , Arash Memarnejadian 4 , Mahdi Assmar 5 , Farzin Roohvand 6
1 - Department of microbiology, Islamic Azad University, Lahijan Branch, Lahijan, Iran
2 - Department Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
3 - Department Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
4 - Department Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
5 - Department of microbiology, Islamic Azad University, Lahijan Branch, Lahijan, Iran
6 - NRGB, Pasteur Institute of Iran, Tehran, Iran
Keywords: Hepatitis C Virus, core+1 protein, Huh7 cell,
Abstract :
Background and Objectives: Core+1 or F protein is recently identified to be encoded by hepatitis C virus (HCV) genome. Although functional properties of this protein are not still discovered, core+1 seems to play important roles in viral life cycle and pathogenesis. This study seeks to clone and express HCV core+1 gene in a eukaryotic system with the final aim of evaluating its potential roles in cell signaling pathways and resistance of HCV to therapy. Material and Methods: Core+1 gene corresponding to the amino acids 1-162 was PCR-amplified from the original gene of subtype b that was previously cloned in a prokaryotic vector (14). The amplicon harboring 6xHis-tag at C-terminal was subsequently subcloned in the eukaryotic pCDNA3.1+ vector. The Nhe I / BamHI restriction sites, initiation and stop codons as well as kozak sequences were designed in the primers. Constructed plasmid was verified by restriction enzyme analysis and sequencing reactions. Liver cell line of Huh7 was transfected with this plasmid using electroporation and lipofection techniques. Transfection efficiency was checked by co-transfection of pEGFPN1 plasmid and flowcytometry. Finally expression and localization of core+1 protein was evaluated by means of confocal microscopic technique. Results: Restriction enzyme analysis and sequencing reactions indicated the accuracy of cloning procedure. Flow cytometric analysis showed higher electroporation efficiency for transfection of Huh7 cells, in comparison with lipofection method. Finally, results of confocal microscopic microscopy using anti-His antibody confirmed the transcription and expression of core+1 protein in Huh7 cells. Conclusions: Our study resulted in the eukaryotic expression of core+1 protein in Huh7 cells and provided an appropriate tool for future studies on the potential interference of core+1 with intracellular signaling pathways and cellular protein interaction.
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