Study On Hepatitis C Virus NS3 Serine Protease And Its Peptide Inhibitors

Hepatitis C is a serious chronic infectious disease that affects about 200 million people worldwide and hepatitis C virus (HCV) is its etiological agent. But no anti-HCV vaccine or specific drugs are available today. HCV non-structural protein 3 (NS3) serine protease which spans 180 N-terminal amino acids of NS3 protein is a multifunctional enzyme. Thus NS3 protease is regarded as a key target to screen specific inhibitor against HCV. In this dissertation, a multi-site inhibitory peptide is designed on the basis of specific substrate sequences of NS3 protease and expressed in prokaryotic cells, and some peptides against this protease are screened using phage surface display peptide technique in order to develop specific anti-HCV drugs. Method: (1) NS3 protease gene and single chain NS3/4A gene were both subcloned into pQE-30 and expressed in E. coli strain M15. The recombinant NS3 and scNS3/4A proteins were purified using ion exchange chromatography. (2) pPICZaA/NS3 and pPICZαA/NS3/4A plasmid were constructed and NS3 protease was expressed in Pichia pastoris (strain GS115) transfected with recombinant plasmids.(3) A NS5A-B fragment containing a cleavage site of NS3 protease was expressed in E. coli strain HB101 with pBVIL/NS5A-B and this NS5A-B can be used as the substrate in an in vitro system when incubated with the recombinant NS3 protease. (4) A multi-site competitive peptide inhibitor is designed with a series of natural substrates' consensus gene sequences gained by Blast analysis online. And then the designed gene was chemically synthesized, cloned, and expressed in prokaryotic cells.(5) The inhibitory peptides...