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The Study on Template Selection in Genomic Replication of Hepatitis C Virus

Author: YeLi
Tutor: YeLinBai
School: Wuhan University
Course: Microbiology
Keywords: Hepatitis C virus HCV genome replication Template selective RNA-dependent RNA polymerase (RdRp) NSSB protein RNA synthesis of cis-acting elements
CLC: R373
Type: PhD thesis
Year: 2005
Downloads: 124
Quote: 1
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Abstract


DNA and RNA is the genetic material, DNA replication mechanisms of people already have a more thorough understanding unclear, but the RNA replication. RNA replication is specific for the virus, the use of positive-strand RNA viruses conduct RNA replication has great significance in terms of the basic theory of molecular biology and genetics, but also to enhance the understanding of RNA viruses, and provide new antiviral therapy policy. Hepatitis C virus (hepatitis C virus, HCV) is enveloped positive strand RNA viruses, the genome length of about 9.6kb, from 5 'coding region and 3' coding region, and a large open reading frame (ORF) of one kind having . ORF encodes an approximately 3000 amino acid residues consisting of poly-protein precursor, after the cutting of the protease encoded by the host protease and HCV virus structural proteins and non-structural proteins, wherein the non-structural protein NS5B has RNA-dependent RNA polymerase ( RNA-dependent RNA polymerase, RdRp) activity, is a key enzyme of the HCV genome replication. Of HCV genome replication and is similar to other positive strand RNA viruses, the first to the positive strand RNA as template to synthesize a complementary negative-strand RNA, and then the newly synthesized negative strand RNA as a template the synthesis of progeny positive-strand RNA, therefore agreed that HCV positive -stranded RNA and the negative-strand RNA 3 'end sequences to play an important role in starting viral genome replication of RNA synthesis. HCV genome replication asymmetric replication: the number of positive strand RNA generated than the negative-strand RNA much more, the former is 10-100 times that of the latter, this phenomenon shows that the HCV genome replication of HCV positive and negative strand RNA template selectivity is very strong. The template selective mechanism is unclear. This thesis uses the purified HCV NS5B protein and HCV positive strand minus strand 3 'end of RNA, case study NS5B using two template for RNA synthesis in vitro conditions, to explore the mechanism of HCV genomic template selective. HCV NS5B gene was cloned into the prokaryotic expression vector pET-His, was transformed into E. coli BL21 (DE3) for expression. In order to improve the soluble expression of the protein, the NS5B C-terminal hydrophobic 21 amino acid deletions, under certain culture conditions, a soluble protein after IPTG induction. Further by histidine affinity chromatography purified protein was determined by Western blot analysis of HCV NS5B protein. RdRp reaction corresponding to the HCV positive the negative strand RNA3 'terminal sequence of the DNA sequence cloned into an in vitro transcription plasmid pGEM3Zf (), prepared by in vitro transcription of HCV positive strand, negative strand 3' end of RNA was, respectively, as the RNA template, by Northern blot detection product synthesis and RT-PCR was not detected to the positive strand RNA as a template, the negative-strand RNA product, while the negative-strand RNA as a template, can be synthesized by a full-length positive strand RNA product; further mixing of the two template , template competition experiments, the positive strand RNA mold

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)
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