الفهرس 
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Abstract
Summary
Hepatitis C Virus was identified in 1989 as the major cause of most cases of non-A, non-B hepatitis (NANBH). HCV belongs to the Flaviviridae family, genus Hepacivirus. It causes acute and chronic liver disease in humans, which often leads to cirrhosis and hepatocellular carcinoma (HCC).
Hepatitis C is a positive-sense-single-stranded RNA virus having a genome of approximately 9600 nucleotides in length. The genome consists of an open reading frame (ORF) flanked by 5’ and 3’ untranslated region (UTR). Both 5’ and 3’ untranslated region have highly conserved RNA structures essential for polyprotein translation and genome replication. Translation of HCV genome leads to a polyprotein of approximately 3000 amino acids that is co- and post-translationally processed by host and virus-encoded proteases in association with the endoplasmic reticulum (ER) to generate the individual mature forms of the viral proteins. The structural proteins include core protein, envelope 1 (E1) and envelope 2 (E2), which form the viral particle. The structural region is followed by the P7 polypeptide and then the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B). HCV is classified in to 6 main genotypes, each of which is further divided into many subtypes. Genotype 4a represents over 90% of cases in Egypt.
The aim of this study is to express selected Hepatitis C genotype 4a peptides and identify their immunodominant epitopes. This is part of a larger study to construct an epitope map for type 4a HCV to aid in the development of more effective reagents and a protective vaccine.
Five peptides comprising 140-202 amino acids from E2/NS2 junction, NS2/NS3 junction, NS3 and NS5A/NS5B junction regions were selected for this study. These selected DNA segments were amplified by PCR then the amplified products were initially cloned in a TA vector, digested with appropriate restriction enzymes and subcloned in the prokaryotic expression vector, pGEX-4T-1. Five peptides were expressed in E. coli TOP10 as fusion proteins with the 26 kDa GST, in the presence of IPTG. The cells were lysed by sonication in order to isolate the epressed proteins. Fusion proteins were purified using affinity purification over glutathione cellulose columns. The expressed proteins were then analyzed on 10% SDS-PAGE. The expected molecular weight was 48 kDa approximately, for peptides 1-5. However, the actual molecular weights were found to be 35, 49, 60, 55 and 50 kDa approximately for peptides 1-5 respectively.
The immunoreactivities of the five expressed fusion peptides and GST were then tested against anti-GST, normal sera and twenty different sera from HCV4a- infected patients using Dot blot analysis, Western blot analysis and ELISA.
Glutathione-S-Transferase (GST) control protein showed immunoreactivity towards anti-GST primary antibody but not towards either normal serum or sera from HCV infected patients, while the five fusion proteins showed immunoreactivity towards anti-GST primary antibody but not to normal serum. All five peptides were also well recognized by HCV infected sera. Peptide # 3 exhibited strong immunoreactivity towards the largest number of sera while peptide # 5 was the least immune responsive.