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研究生: 郭佳貞
Chia-chen Kuo
論文名稱: 銀奈米吸附於特定肽腱對於HIV-1蛋白酶之抑制效果
The Inhibition of HIV-1 Protease by Specific Substrate Bound with Silver Nanoparticles
指導教授: 李篤中
Duu-jong Lee
口試委員: 劉志成
Jhy-chern Liu
李懷特
Chris Whiteley
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2014
畢業學年度: 102
語文別: 英文
論文頁數: 94
中文關鍵詞: 銀奈米HIV-1愛滋病HIV-1 蛋白酶
外文關鍵詞: silver nanoparticle, HIV-1, AIDS, HIV-1 protease
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現今常用於治療愛滋病的治療法為高效抗逆轉錄病毒治療,又稱雞尾酒療法。根據報告指出雞尾酒療法近年已見成效,使愛滋病感染者的數量和愛滋病的死亡率皆下降。然而,雞尾酒療法將會面臨一個不可避免的難題,抗藥性的產生。此療法之療效將會因抗藥性的散佈而逐漸下降,因此研究發展出一個不會產生抗藥性的治療方法是不可或缺的。
銀奈米粒子有特別的機制用來抑制細菌,此機制不會產生抗藥性,所以銀奈米粒子近期也被用於病毒上以測試其抑制病毒的效果。已有報告指出可由體外實驗得到銀奈米粒子可以抑制愛滋病蛋白酶。然而,銀奈米粒子對於愛滋病蛋白酶並無特定性,若將銀奈米粒子置於體內,有可能會因為其附著於含有硫原子的肽腱而導致抑制病毒的效果下降。因此,找到一個有特定性的肽腱,其可以吸附銀奈米粒子,並且與愛滋病蛋白酶作用,而進一步抑制愛滋病帶白酶的活性是很重要的。
擁有愛滋病蛋白針對的切割位置和不同位置的含硫胺基酸的合成肽腱組被用來找出其與愛滋病蛋白酶和銀奈米粒子彼此之間的反應關係。可藉由合成肽腱組與愛滋病蛋白酶之間的反應求出愛滋病蛋白酶的酵素活性。可將其與已附著銀奈米粒子的肽腱和愛滋病蛋白酶之間的反應結果作比較,求得已附著銀奈米粒子的肽腱對於愛滋病帶白酶的抑制效果。另外,紅位移可作為銀奈米粒子附著於硫原子上的證據。比較後,可知已附著銀奈米粒子的肽腱確實能對愛滋病蛋白酶具有抑制作用。


Even though the common therapy for AIDS, Highly Active Anti-Retroviral Therapy (HAART) has a reasonable level of success against HIV infections and HIV-related death, the problem of drug resistance from the HIV still persists. Consequently there is an ongoing need to develop new strategies and novel drugs against this.
It has been reported that silver nanoparticles (Ag NPs) with their unique anti-bacterial properties can not only overcome this drug resistance but can also inhibit the HIV protease (HIV-1 PR) in vitro. Since these particles, however, are not specific to HIV-1 PR, a search was undertaken for a peptide that is not only specific for the protease but can interact with silver nanoparticles as well and lead to enzyme inhibition. Since earlier studies suggested that silver nanoparticles have a strong attraction to sulfur, specific peptides containing one, or more, cysteine amino acids and an aromatic-hydrophobic cleavage site were synthesized.

ACKNOWLEDGMENT IV ABSTRACT V 摘要 VI TABLE OF CONTENTS VII LIST OF FIGURES X LIST OF TABLES XIII CHAPTER 1 – INTRODUCTION 1 1-1 Background 1 1-2 Research Goals 2 CHAPTER 2 - LITERATURE REVIEW 3 2-1 Human Immunodeficiency Virus (HIV) 3 2-1-1 Current Situation of HIV Prevalence 3 2-1-2 Types and Structure of HIV 4 2-1-3 Life Cycle of HIV 5 2-1-4 HIV-1 Protease 7 2-1-5 Inhibition of HIV-1 Protease 10 2-2 Silver Nanoparticles in Biomedical application 14 2-2-1 Antibacterial 14 2-2-2 Antiviral against HIV-1 16 2-2-3 Medical Applications 18 2-2-3-1 Wound Dressing 18 2-2-3-2 Getting in Medical Supplies and Devices 18 2-2-4 Health Concerns 19 2-3 Silver Nanoparticles synthesis 20 2-3-1 Mechanism of Wet Chemical Synthesis 21 2-3-2 Metal Precursor and Reducers 23 2-3-3 Stabilizers 25 2-4 Analysis of Silver Nanoparticles 27 2-4-1 Zeta Potential 27 2-4-2 Dynamic Light Scattering (DLS), Zetasizer 29 2-4-3 Ultraviolet-Visible (UV-vis) Spectroscopy 30 2-4-4 Transmission Electron Microscopy (TEM) 31 2-4-5 pH measurement 31 2-5 Synthesized Peptides 33 2-5-1 Substrate Specificity of HIV-1 Protease 33 2-5-2 Adsorption of Substrate with sulfur atom to Silver Nanoparticles 34 CHAPTER 3 - MATERIALS AND METHODS 36 3-1 Materials 36 3-1-1 Experimental Chemicals 36 3-1-2 HIV-1 Protease 38 3-1-3 Synthesized Peptide 38 3-2 Methods 40 3-2-1 Synthesis of Silver Nanoparticles (Ag NPs) 40 3-2-1-1 Preparation for Experimental Materials of Ag NPs Synthesis 40 3-2-1-2 Steps of Ag NPs Synthesis 41 3-2-2 Silver Nanoparticles Characterization 41 3-2-2-1 Zeta Potential 42 3-2-2-2 Dynamic Light Scattering (DLS), Zetasizer 42 3-2-2-3 Ultraviolet-Visible (UV-vis) Spectroscopy 43 3-2-2-4 Transmission Electron Microscopy (TEM) 43 3-2-2-5 pH measurement 43 3-2-3 EDANS/HEPES/DMSO Characterization 44 3-2-3-1 Ultraviolet-Visible (UV-vis) Spectroscopy 44 3-2-4 Interaction of Ag NPs with EDANS/HEPES/DMSO 44 3-2-4-1 Ultraviolet-Visible (UV-vis) Spectroscopy 45 3-2-5 HIV-1 Protease (PR) Characterization 45 3-2-5-1 pH measurement 45 3-2-6 Synthesized Peptides 46 3-2-6-1 Ultraviolet-Visible (UV-vis) Spectroscopy 46 3-2-6-2 pH measurement 46 3-2-7 Interaction between Ag NPs, Synthesized Peptides and HIV-1 PR 47 3-2-7-1 Ultraviolet-Visible (UV-vis) Spectroscopy 47 3-2-7-2 pH measurement 47 CHAPTER 4 - RESULTS AND DISCUSSIONS 49 4-1 Silver Nanoparticles Characterization 49 4-2 Reaction between Ag NPs with EDANS/HEPES/ DMSO 53 4-3 Synthesized Peptides Characterization 57 4-3-1 Results about Synthesized Peptide No.1 57 4-3-2 Results about Synthesized Peptide No.2 58 4-3-3 Results about Synthesized Peptide No.3 59 4-3-4 Results about Synthesized Peptide No.4 61 CHAPTER 5 - CONCLUSION 74 REFERENCES 76

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