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Screening of Proteins Binding Gene2 Tranactivated by Hepatitis C Virus NS3 Protein from Liver cDNA Library by Yeast Two-hybrid Technique

Author: HanLi
Tutor: ZhangLianFeng
School: Zhengzhou University
Course: Internal Medicine
Keywords: NS3TP2 Yeast two-hybrid Expression Fat metabolism Protein metabolism
CLC: R346
Type: Master's thesis
Year: 2007
Downloads: 73
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Abstract

First, the background and purpose of the hepatitis C virus (HCV) is a Flaviviridae, can cause chronic hepatitis, cirrhosis and liver cancer, currently the world's 170 million people are infected, only treatment with interferon and ribavirin, and limited efficacy . For the the HCV molecular mechanisms clarify important prerequisite for antiviral treatment of hepatitis C and related diseases and eventually overcome. HCV genome transcription and translation of a polypeptide precursor, and then the effect of the signal peptidase and protease cleavage of the 10 proteins, including Core, E1, E2, P7, NS2, NS3, NS4A, NS4B, the NS5A, the NS5B. Which encodes 631 amino acids, the non-structural protein NS3 of the size of approximately 70KD is a multifunctional protein, having serine protease nucleotidase triphosphate (NTPase) and RNA helicase (helicase) function, in the HCV replication and expression The product poly cleavage processing of the protein to play an important role. The studies show that the HCV NS3 protein may also be regulated by the trans-activation of transcription of a variety of viral and cellular genes in the cells, plays an important role in the HCV pathogenicity (cancerous) process. Party Xiaoyan using suppression subtractive hybridization technology the (suppression substractive hybridization) and bioinformatics technology (bioinformatics) screening and cloned NS3 protein trans-activation of target genes and found that it is up-regulated genes in addition to genes of known function also included in some of the genes of unknown function. One named NS3TP2 (HCV NS3-transactivated protein 2), bioinformatics techniques to determine the ORF and cloning research, smooth been NS3TP2 the gene coding sequence registered in GenBank, the registration number AY116970 . Expressed in yeast cells in order to further study NS3TP2 biological function of the protein in the liver cDNA library using the yeast two-hybrid screening their protein gene to the role in in HCV pathogenesis of the HCV NS3 protein The study provides new clues. Research methods: application of the yeast two-hybrid system 3, the NS3TP2 gene amplification by polymerase chain reaction (PCR) method to connect into the yeast expression vector pGBKT7 construct the bait plasmid transformed haploid yeast cells AH109 and expression within its extract yeast proteins, dodecyl sodium sulfonate - polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis and then transformed haploid yeast cells of the human liver cell library plasmid pACT2 Y187 blended in the coating X-α-galactose (X-α-gal) auxotrophic medium (SD /-Trp-Leu-His-Ade) on the growth of double filters, extraction of the plasmid of the positive yeast clones, and transformed into E. coli plasmid DNA was extracted , then this positive the library plasmids bait plasmid backcross experiment further screening, sequencing, sequencing results in GenBank bioinformatics analysis. Results: Success to build NS3TP2 protein gene yeast expression vector, Western immunoblot shows NS3TP2 protein expression in yeast cells. Expression products in the cellular memory, molecular weight of about 68 000 kd. After a backcross experiment screened 6 kinds of the combination with NS3TP2 protein gene, including vacuolar protein the 13 homologues A (VSP13A), acyl-coenzyme A synthetase long-chain family members 5 (ACSL5), mitochondrial genes, acyl-CoA A regulatory factor binding protein (r-aminobutyric acid receptor, diazepam binding inhibitor), eukaryotic translation elongation factor alpha 1, ring finger protein 130. Conclusion: 1. Successfully constructed the pGBKT7-NS3TP2 yeast expression plasmid. 2. Successfully expressed in yeast cells for the first time 10 pGBKT7-NS3TP2 fusion protein. 3. Using the yeast two-hybrid technology has successfully screened in the liver cells HCVNS3TP2 protein interacting protein gene. A class of gene and protein metabolism: vacuolar protein 13 homologue A RING finger protein 130, eukaryotic translation elongation factor alpha 1. A class of genes associated with fat metabolism: a member of the family of long-chain acyl-coenzyme A synthetase of 5 (ACSL5) and acyl coenzyme A binding protein (ACBP). 4. NS3TP2 as HCV NS3 trans-activating protein in the metabolism of protein and fat metabolism of the liver cells play a role in lipid metabolism, it may be mediated by NS3 of HCV infected hepatocytes lipid metabolism , played a definite role in the occurrence of HCV infection in patients with fatty liver, and provide new clues for NS3TP2 biological function and the pathogenesis of HCV NS3.

Screening of Proteins Binding Gene2 Tranactivated by Hepatitis C Virus NS3 Protein from Liver cDNA Library by Yeast Two-hybrid Technique

Abstract

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