CDNA Cloning, Expression And Analysis Of Antigenic Reactivity Of NS5A Regions Of HCV

Hepatitis C Virus(HCV) is an enveloped positive single-stranded RNA (9.4-kilobases) virus belonging to the Flaviviridae.The HCV genome encodes a large polyprotein precursor of about 3,000 amino acid residues, and this precursor protein is cleaved by the host and viral proteinases to generate 10 proteins in the following order:NH2-core-envelope(El)-E2-p7-non-structural-protein2(NS2)-NS3-NS4A-NS4B-NS5A-NS5B-COOH..The non-structural 5A(NS5A) protein of hepatitis C virus (HCV) is a 56, 000 MW phosphoprotein (p56), which also exists within infected cells in a hyperphosphorylated form (p58). Its precise functions are not known. NS5A can also bind to single-strand RNA-dependent protein kinase (PKR) and inhibit its antiviral function. An 'interferon - sensitivity-determining region'(ISDR) has recently been postulated in the NS5A protein central region in hepatitis C virus (HCV) genotype 1 b. therefore,we began to study the HCV-NS5A gene as follows:Part I Cloning of the fragment of HCV-NS5A-1U1D geneIn our study ,A gene fragment of HCV-NS5A was cloned by RT-PCR ,PCR wasemployed to gain HCV-NS5A-1U1D gene,then the product of PCR was ligated to thevector PMD-T,which was named PMD-T-lU1D.Identified by restriction enzymedigestion and compairing cDNA sequences of our clones with those of HCV-J ,We canconclude that the gene fragment of HCV-NS5A-1U1D belonged to genotype of 1b ofHCV.Part II Expression of the fragment of HCV-NS5A-1U1D geneHCV-NS5A-1U1D gene was ligated to the expressional vector PQE30,E-coli Ml5 was transformed by the recombinant PQE30-1U1D and induced by IPTG.A molecular weight of 20 10 soluble protein was expressed, which had a poly-histidine (6 his)"tag"at the N-terminal,resulted in simple purification by Ni-NTA affinity chromatography.Part III Analysis of antigenticity of the fragment of HCV-NS5A-1U1D gene The fusion protein containing the peptide encoded by HCV-NS5A-1U1D was synthesized by E.coli M15 with recombinant plasmid.The specific HCV antigenicity of the fusion protein was identified by ELISA.It demonstrated that the purified protein had specific HCV antibody-binding activity .The result indicated that the expressed protein can be used to establish an EIA method for detection of anti-HCV NS5A.