| Hepatitis C virus (HCV) is a single-stranded positive RNA virusbelonging to the Flaviviridae family. The HCV genome contains a 5'and a 3'untranslated regions (UTR) and a single open reading frame (ORF) encoding a polypeptide of about 3,000 amino acids. The polypeptide precursor is cotranslationally and posttranslationally processed by both cellular and viral proteases at the endoplasmic reticulum membrane to yield 10 mature proteins. From 5' to 3', they are C, El, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B protein. HCV is a major cause of chronic hepatitis and has close relationship with liver cirrhosis, and hepatocellular carcinoma worldwide. About 170 millions individuals are infected with HCV that is five times as widespread as infection with the human immunodeficiency virus type 1 (HIV-1). HCV infection also increases the number of complications in persons who are coinfected with HIV-1. The research on pathogenesis of HCV was harmbered by lacking efficient culture sytem. It is not very clear to the molecules involved in HCV recognition, attachment, and cell entry and how thevirus infected liver cells. The two envelope glycoproteins, El and E2, are thought to play pivotal roles at different steps of HCV replicative cycle. There is now strong evidence that they are essential for host-cell entry, by binding to receptor(s) and inducing fusion with a host-cell membrane. The interaction between HCV and its receptors on cell surface induced the configuration of El and E2, which can cause the HCV fused with cell membrane. Then the HCV parcels were interlized and the endosome was formed.One of the initial steps in a viral infection and propagation is the binding of the virus to cell surface molecules. It is one of the main factors that determining virus host specificity, tissue-tropism and pathogenicity. Studies about HCV receptor may help to explain the pathogenicity of HCV and provide new clues for finding new anti-virus methods.It has been found that human SR-BI could bind to the envelope protein E2 of HCV. According to the result that has been published by other researchers presuming that the human SR-BI maybe a novel candidate receptor for the hepatitis C virus. To investigate the role of human SR-BI in cellular entry of HCV, the following work have been done:1 the Prokaryotic expression and purification of HCV glycoproteinsThe genes of envelope proteins E2 and E1E2 of HCV were amplified from pBRTM/HCVl-3011, a plasmid containing the cDNA of HCV's ORF, by PCR and were cloned into plasmid pGEM-Teasy. The plasmid containing the gene of envelope proteins E2 or E1E2 was identified by clone-PCR or enzyme digestion and sequenced. Prokaryotic expression plasmid of the envelope proteins E2 and E1E2of HCV was constructed by cloning the gene of E2 or E1E2 into pET28(a), a prokaryotic expression plasmid belongs to pET families. The prokaryotic expression plasmid of E2 or E1E2 was transformed into E.coli, and the expression of protein E2 or E1E2 was induced by IPTG. The protein E2 or E1E2 was purified by Ni-NT agarose column respectively. The results of ELISA and Western blot assay suggested that all of them have the biological activities. This work set a molecular biology basis for preparing the polyclonal antibody to two proteins and constructing the eukaryotic expression vector of E2 or E1E2.2. Preparation of polyclonal anti-sera to HCV envelop proteins New Zealand rabbits were immunized with the glycoprotein E2 and E1E2 from HCV. ELISA and Western bloe assay were used to detect the biological activities and titer of the polyclonal anti-sera. When they are diluted to 1:1280, the polyclonal anti-sera are still having the biological activities. The polyclonal anti-sera supply an effective tool for more researches on HCV glycoproteins interacting with their receptors.3 Eukaryotic expression and puification of HCV glycoproteins The genes of envelope proteins E2 and E1E2 of HCV were amplified by PCR and were cloned into plasmid pBluescript II SK(+)-hC y 1. The genes fused envelope proteins E2 or E1E2 with hu... |
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