الفهرس 
Review of literature
Selection of positive transformantsOnly 14 pages are availabe for public view
Abstract
Hepatitis C virus represents a serious worldwide health
care 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.
Over the past 10 years, the growth in scientific
understanding of the HCV life cycle and new technologies to
measure HCV replication played a critical role in the
development of a number of models to study the HCV life
cycle and assess the potency of drugs that disrupt it. Given its
essential roles in viral polyprotein processing and immune
evasion, HCV NS3/4A protease is a prime target for antiviral
chemotherapy.
The aim of the present study was to establish in vivo cellbased
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.HCV NS32-181 protease cDNA fragment was PCR
amplified and then cloned into pGEM-T vector followed by
transformation into bacterial competent cells. HCV NS3/4A
cDNA fragment was then subcloned into the pGEX-4T-1
expression vector.
Native as well as mutant cleavage sites to NS3/4A
protease were cloned in frame into β-galactosidase gene
of pGEM-T cloning vector. The target specificity of
HCV NS3/4A was evaluated by co-expression of β-
galactosidase containing the protease cleavage site with
NS3/4A protease construct in bacterial cells. The
activity of β-galactosidase was colorimetrically
estimated in the cell lysate using ortho-nitro phenyl β-Dgalactopyanoside
(ONPG) as a substrate.
The efficiency of the established system was
monitored using series of computer-designed
chemically-synthesized compounds (7a [BE113], 7b
[BE114], 8a [BE115] and 8b [BE116]) that were
designed against NS3/4A protease from HCV genotype
4a, PMSF was used as a positive control and DMSO as a
negative control The HCV3/4A protease enzyme has a proteolytic
activity towards its native substrate encoded by β-gal HCV
5A5B construct, while had no remarkable proteolytic
activity towards its mutant substrate encoded by β-gal mt.
HCV 5A5B construct. The compounds BE114 (7b) and
BE115 (8a) had moderate inhibitory efficacy against HCV
NS3/4A protease, while compounds BE113 (7a) and BE116
(8b) had no remarkable inhibitory effects on the protease
enzyme.
The developed system represents a simple, rapid, and
highly specific assay that can be used to monitor the
activity of the HCV NS3 serine protease, and it has the
potential to be used for screening specific inhibitors.
Recommendation:
The developed system can be used in the future to transfect
mammalian cells in cell culture in order to monitor the
efficacy of new inhibitors that will be designed against
HCV NS3 protease.