RESUMO
Background: Hepatitis C virus (HCV) represents a serious worldwide healthcare problem. No protective vaccines against HCV have been developed yet due to the fact that HCV is rapidly mutable, allowing the virus to escape from the neutralizing antibodies. Understanding of HCV was initially hampered by the inability to achieve viral replication in cell culture. Given its essential roles in viral polyprotein processing and immune evasion, HCV NS3/4A protease is a prime target for antiviral chemotherapy. We aimed to establish in vivo cell-based assay system for monitoring the activity of NS3/4A protease from HCV genotype 4a, the predominant genotype in Egypt, and the Middle East. Furthermore, the developed system was used to evaluate the inhibitory potency of a series of computer-designed chemically-synthesized compounds against NS3/4A protease from HCV genotype 4a. Materials and Methods: Native as well as mutant cleavage sites to NS3/4A protease were cloned in frame into ?-galactosidase gene of TA cloning vector. The target specificity of HCV NS3/4A was evaluated by coexpression of ?-galactosidase containing the protease cleavage site with NS3/4A protease construct in bacterial cells. The activity of ?-galactosidase was colorimetrically estimated in the cell lysate using orthonitro phenyl ?-D-galactopyanoside (ONPG) as a substrate. Results and Conclusions: We successfully developed an efficient cell-based system based on the blue/white selection of bacterial cells that are able to express functional/nonfunctional ?-galactosidase enzyme.
RESUMO
Background: Hepatitis C virus (HCV) is characterized by a high degree of nucleotide sequence variability between genotypes. This variability extends to functional and immunological determinants. Serological tests using antigenic segments derived from the HCV polyprotein have been used for the diagnosis of HCV infection. However, available diagnostic Kits do not necessarily take type variability into consideration and are not optimized for HCV genotype 4a (HCV4a), the predominant genotype in Egypt. Aim: The aim of this study was to express some HCV4a-derived polypeptides in order to identify those with immunodiagnostic utility. Materials and Methods: Six sequential/overlapping genomic segments encoding 100–266 amino acid peptides from the core (peptide 1), envelope 1 (E1; peptide 2), envelope 2 (E2; peptides 4, 5, and 6), and E1/E2 (peptide 3) regions of the HCV4apolyprotein were selected for in vitro expression as glutathione S-transferase-fusion proteins. The immunoreactivity of the expressed peptides was evaluated against sera from HCV-infected/uninfected individuals using dot blot, western blot, and enzyme-linked immunosorbent assay. Results: The expressed polypeptides were recognized by HCV-infected sera from 20 patients, while showing no immunoreactivity toward uninfected serum. Peptide 1 derived from the core protein was found to be the most immunoreactive. Conclusion: Expressed polypeptides hold good potential for use in the development of improved HCV immunodiagnostics.