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研究生:陳氏雨雯
研究生(外文):Nhung Thi Tuyet Tran
論文名稱:C型肝炎之蛋白酶的表現與定性分析及其抑制劑之篩選
論文名稱(外文):Expression and Characterization of Tailor-Made HCV Proteases and Screening for Its Inhibitors
指導教授:黃贊勳
指導教授(外文):Hwang,Tzann-Shun
口試委員:李佳音許德賢黃贊勳
口試委員(外文):Lee, Chia-YinSheu, Der-ShyanHwang,Tzann-Shun
口試日期:2013-06-26
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:124
中文關鍵詞:C型肝炎之蛋白酶的
外文關鍵詞:HCV protease
相關次數:
  • 被引用被引用:0
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Hepatitis C virus (HCV) infection is a global health burden with over 180 million people infected worldwide and without vaccine available currently. HCV genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. The HCV NS3 protein is an essential protease for viral polyprotein processing. For full activity, the NS3 protease requires a NS4A protein as a cofactor. NS4A/NS3 protease cleaves the polyprotein at four specific regions NS3-NS4A, NS4A-NS4B, NS4B-NS5A and NS5A-NS5B junctions. NS3 and its recombinant protease are regarded currently as a potential target for anti-viral drugs. Thus, specific inhibitor of its enzyme activity is highly important.
In this study, recombinant protein NS4A/NS3 was constructed by fused NS3 with NS4A peptide by different linkers (GSGS and GG). NS4A/NS3 with GSGS was found to have higher activity than NS4A/NS3 with GG and NS3con + Pep4A (wild type), however, wild type was more stable than fusion proteases. The best assay conditions were established for all tailor-made HCV proteases. To screen inhibitors against NS3 protease, software of molecular docking model (Autodock version 4.2) was used to evaluate the free energy after NS4A/NS3 protease was docked with ligands from chemical library (chemical compounds bank). After checking the docking score of 50,000 peptide bond-containing compounds, three compounds with free energy less than -10 Kcal/mol were obtained (Candidate 1, 2 and 3). A natural inhibitor, hexapeptide DEMEEC, was used to examine the inhibitory potency of three constructs. The kinetic and inhibitory parameters, Km, IC50 and Ki, of NS3con + Pep4A against hexapeptide were found to be 4.3 µM, 2.5 µM and 0.51 µM, respectively. That of NS4A-GSGS-NS3 against hexapeptide were 2.45 µM, 4.23 µM, 1.04 µM, respectively, and that of NS4A-GG-NS3 against hexapeptide were 3.71 µM, 4.9 µM, 2.54 µM, respectively. One of candidates, Candidate 1, was found to have high inhibition effect against NS4A/NS3 protease. The inhibitory parameters, IC50 and Ki, of NS3con + Pep4A against Candidate 1 were found to be 25.4 µM and 5.04 µM, respectively; that of NS4A-GSGS-NS3 against Candidate 1 were 61.1 µM and 7.88 µM, and that of NS4A-GG-NS3 against Candidate 1 were 87.9 µM and 6.89 µM, respectively. The Lineweaver–Burk plot of inhibitory kinetics showed that candidate 1 acted as a competitive manner, which suggested that it competes with substrate to bind into active sites. Interaction between Candidate 1 and NS3 protease domain simulated by PyRx software that showed binding affinity of candidate 1 with His57 and Ser139 (catalytic triad). The inhibition activity of Candidate 1 is not good enough, but it could be further modified on its chemical structure to improve the inhibitory activity and to be a better inhibitor.

ABSTRACT iii
ABBREVIATION vii
LIST OF TABLES ix
LIST OF FIGURES x
CHAPTER ONE INTRODUCTION 1
1.1 Introduction 1
1.2 Hepatitis C virus 3
1.2.1 HCV genome structure 3
1.2.2 HCV replication 6
1.3 Hepatitis C virus recombinant protease (NS4A/NS3) 6
1.4 Inhibitors of the HCV NS4A/NS3 serine protease 11
1.4.1 Peptide NS4A/NS3 protease inhibitors 11
1.4.2 Non-peptide NS4A/NS3 protease inhibitors 17
1.5 Hepatitis C virus (HCV) current treatment 17
1.6 Molecular simulation docking modeling 18
1.7 Enzyme kinetics and inhibition 19
1.8 Research Aim 23
CHAPTER TWO MATERIALS AND METHODS 24
2.1 Experimental materials 24
2.1.1 Bacterial strains, expression vectors and reagents 24
2.1.2 Chemical compound library, enzyme structure and docking software 24
2.2 DNA manipulation for HCV protease construction 25
2.2.1 DNA manipulation 25
2.2.2 DNA transformation 26
2.2.3 PCR amplification 27
2.2.4 DNA recovery 28
2.2.5 TA vector cloning and blue and white screening 28
2.2.6 Expression vector cloning 29
2.3 Expression and optimization of HCV single chain protease 30
2.3.1 Expression and purification of HCV single chain NS3 protease 30
2.3.2 Optimization of enzyme activity 31
2.4 Molecular simulation for docking 33
2.4.1 Preparation of the 3D enzyme structure for docking 33
2.4.2 Preparation of chemical compounds library 33
2.4.3 Docking chemical compounds library to 3D enzyme structure 33
2.5 Enzyme Assay of inhibitor candidates 36
2.6 Kinetics analysis of HCV protease inhibitor 37
CHAPTER THREE RESULTS AND DISCUSSION 39
3.1 Construction of recombinant HCV protease (NS4-GSGS-NS3 and NS4-GG-NS3) 39
3.2 Expression, purification and characterization of the recombinant HCV protease 41
3.3 In silico molecule modeling to screening HCV protease inhibitors by using Autodock software 45
3.4 Kinetic analysis of HCV recombinant protease activity 46
3.5 Inhibiton assay of HCV recombinant protease 48
3.6 Analyzing docking results with PyMol software 50
CHAPTER FOUR CONCLUSION 52
REFERENCE 55
APPENDICES 101


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