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研究生： 林彥甫
Yen-Fu Lin
論文名稱： 胃幽門螺旋桿菌26695結構基因體-HP0404蛋白質結構與功能的探討
Structural genomics of H. pylori 26695-Solution structural and functional studies of protein HP0404 from Helicobacter pylori
指導教授： 程家維
Jya-Wei Cheng
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生物科技研究所
Biotechnology
論文出版年： 2005
畢業學年度： 93
語文別： 中文
論文頁數： 127
中文關鍵詞： 幽門螺旋桿菌 、蛋白質激酶C抑制劑 、超高速離心機 、旋光儀 、螢光儀 、感應偶合電漿質譜儀 、核磁共振
外文關鍵詞： Helicobacter pylori, protein kinase C inhibitor, Analytical ultracentrifuge, Circular Dichroism, Fluorescence Spectrometer, ICP-MS, NMR
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    • 近幾年來，由於結構基因體學蓬勃發展，基因序列資訊結合了最新的分子與結構生物學，使得大家對結構基因有更深一層的認識與探討。幽門螺旋桿菌（Helicobacter pylori, HP）是一個格蘭氏陰性菌、成長慢、具有鞭毛及螺旋外狀的細菌，它會引起很嚴重的胃潰瘍與十二指腸的疾病，幾乎全世界有一半的人口均受此菌感染。由於目前科學家們對於幽門螺旋桿菌的基因體序列所產生之蛋白質了解甚少，因此，我們選擇了幽門螺旋桿菌作為整個結構基因體計畫的主角。
    這篇論文主要是研究幽門螺旋桿菌基因體中的一個蛋白質-HP0404，利用胺基酸序列比對的結果，推測其生物活性可能為PKC的抑制劑。藉由旋光儀偵測HP0404的二級結構和蛋白質穩定性，顯示HP0404是一個熱穩定性蛋白。另外在生物活性研究方面，使用螢光儀偵測蛋白質與金屬離子的鍵結能力，並且以ICP-MS將蛋白質內的金屬離子作定量分析，印證了HP0404對Zn2+具有專一性的鍵結作用。此外，利用非放射性PKC活性偵測實驗，測試出HP0404的確具有抑制PKC活性的功能。蛋白質結構方面，以NMR技術分析HP0404和HP0404 C14A的蛋白質結構之差異性。不但如此，X-Ray技術的應用也在嘗試研究中，以點晶方式將HP0404蛋白質培養成晶體，目的就是希望藉由不同方式研究HP0404蛋白質的分子結構。

    In recent years, the structural genomics has been studied and developed thoroughly. Genomic sequencing information has combined the latest molecular and structural biology; hence scientists are able to move one step further in the structural genomics. Helicobacter pylori is a Gram-negative, microaerophilic, and slow-growing bacterium with flagellum and spiral appearance. It causes serious gastric ulcer and duodenum-associated diseases, and almost half population of the world has been infected by this bacterium. Since scientists have little understanding in the proteins produced by Helicobacter pylori genomics so far, we have chosen this bacterium as the major subject of the structural genomic project.

    The aim of this thesis is to investigate a protein called HP0404 from Hlicobacter pylori genomics. The results from amino acid sequence alignment inferred that its biological activity might be inhibition of PKC. The secondary structure and stability of the protein were determined by circular dichroism, and it revealed that HP0404 is a heat stable protein. In addition, fluorescence spectrometer was used to detect the binding ability between the protein and metal ions, and the quantitative analysis of the metal ions within the protein was performed by ICP-MS, thus it is shown that HP0404 has specific binding with Zn2+. Furthermore, the non- radioactive method was applied to demonstrate that HP0404 could effectively suppress the PKC activity. The difference between the protein structures of HP0404 and HP0404 C14A was examined by NMR. Moreover, we attempted to use X-ray technology to study the protein structure of HP0404. The hanging-drop method was applied to crystallize HP0404, and the structure would be determined by X-ray. The purpose was to study the molecular structure of HP0404 in a different way.

    Abstrate……………………………………………….… ..1 中文摘要……………………………………………….... ..2 第一章 序論（Introduction）….……….………..…… 8 第一節 結構基因體學(structural genomics)……………… …8 第二節 幽門螺旋桿菌(HelicobacterPylori)…………………8 第三節 HP0404蛋白質的目標基因篩選……………………………11 第四節 PKCI(Protein Kinase C Inhibitor)的功能及結構研究12 第五節 HP0404與PKC inhibitor的關係………… …...14 第六節 HP0404蛋白質的研究目標…………………… ….15 第二章 材料與方法（Material and Methods ）...… 16 第一節 Cloning HP0404………………………………………….16 1.1 載體(vecter) - pETBlue-2 Vector……………………...…16 1.2 引子(primers) 合成………………………………….... 17 1.3 聚合酶鏈鎖反應(PCR，polymerase chain reaction)…………17 1.4 去氧核酸電泳(DNA electrophoresis)……………………….18 1.5 PCR產物的膠上純化(Viogene Gel-M Gel extraction system)18 1.6 限制酵素反應(restriction enzyme reaction)……………….19 1.7 接合作用(ligation)………………………………………......20 1.8 轉殖細胞之製備(competent cell preparation)………………21 1.9 細菌轉殖(bacterial transformation…………………………23 1.10 蛋白質表現量測定（protein expression test）……………27 第二節 定點突變法(Site-directed mutagenesis)……………27 第三節 非變性的聚丙烯醯胺凝膠的製備…………………………28 3.1 電泳原理……………………………………………………28 3.2 非變性的聚丙烯醯胺凝膠………………………………. 29 3.3 凝膠製備方式………………………………………………… 33 第四節 蛋白質的表現與純化(protein expression and purification)……. .31 4.1 蛋白質大量表現培養(large-scale expression culture)……31 4.2 蛋白質純化(protein purification)………………………….32 4.3 SDS-PAGE分析(SDS-PAGE analysis)……………………33 第五節 光電比色儀（Spectrophotometer）……………………34 5.1 實驗原理………………………………………. ……………34 5.2 實驗方式……………………………………………….… …34 第六節 超高速離心機XL-A(Analytical ultracentrifuge)……35 6.1 實驗原理……………………………………………...… …35 6.2 實驗方法……………………………………………..……. …37 6.3 實驗樣品製備………………………………………..…….……37 第七節 質譜儀（Mass Spectrometer）…………………………37 第八節 旋光儀 (Circular Dichroism )…………………… …38 8.1 旋光儀光譜分析 (circular dichroism spectrum analysis).38 8.2 熱變性實驗（Thermal Denature Experiments）…………….39 8.3 化學變性實驗(chemical denaturation experiments)………40 8.4 旋光儀 (circular dichroism) 樣品製備…………... …40 第九節 螢光光譜儀（Fluorescence Spectrometer）………..41 9.1 螢光光譜分析(Fluorescence spectrum analysis)…………41 9.2 基本原理………………………………………………..… 41 9.3 樣品製備…………………………………………..……….…43 9.4 實驗方式…………………………………………………….43 第十節 感應偶合電漿質譜儀（ICP-MS）…………………...…44 10.1 儀器簡介……………………………………………… …44 10.2 基本原理………………………………………….… …44 10.3 HP0404蛋白質的培養………………………………….…46 10.4 樣品製備………………………………………………….… 47 10.5 計算實驗結果…………………………………………….… …47 第十一節 PKCI(protein kinase C inhibitor)的活性偵測實驗…48 11.1 實驗原理…………………………………………….…………48 11.2 實驗材料…………………………………………………….…49 11.3 實驗步驟……………………………………………………….50 11.4 磷酸化peptides的定量…………………………………………51 11.5 酵素活性的計算…………………………………………….…52 第十二節 核磁共振(NMR)分析………………….……………………55 12.1 樣品製備…………………………………….…………………55 12.2 核磁共振歷史和基本原理……………….………………..…55 12.3 核磁共振NMR experiments……………………………….…..60 12.4 氫氘交換與骨架動力學光譜實驗(Dynamics experiments) 68 12.5 利用TALOS 來預測phi 和psi backbone torsion angles……68 12.6 蛋白質結構的計算………………………………………...…68 12.7 核磁共振光譜處理與分析 (Data processing and analysis)70 第十三節 結晶實驗(Crystallization experiments)……….……71 13.1 蛋白質結晶原理（Crystallization of protein）………….71 13.2 蛋白質結晶條件與結晶方式………………………… …72 第三章 實驗結果與討論（Result and Discussion）…73 第一節 HP0404的目標基因篩選與二級結構預測………….……73 第二節 HP0404蛋白質的表現與純化………………………….…74 第三節 分析HP0404蛋白質之分子量………………………….…75 3.1 分析級超高速離心機（Analytical ultracentrifuge analysis）75 3.2 質譜儀（Mass）………………………………………........76 第四節 HP0404蛋白質物性與化性的研究………………………76 4.1 HP0404在不同pH環境中對蛋白質結構之影響…………..… 76 4.2 熱變性實驗………………………………………………... 77 4.3 化變性實驗…………………………………….…………..…77 第五節 HP0404蛋白質與金屬離子的鍵結作用………….……78 5.1 HP0404與鋅離子的鍵結…………………………………… 78 5.2 HP0404與其它金屬離子的鍵結……………………………… 79 5.3 利用ICP-MS 分析HP0404蛋白質中的Zn2+ ………………… 79 第六節 偵測HP0404蛋白質對PKC之抑制能力………..…… 80 第七節 HP0404蛋白質的結構分析………………… ………….81 7.1 HP0404 one-dimensional NMR實驗………………………… 81 7.2 HP0404與HP0404 C14A的two-dimensional NMR實驗……… 81 7.3利用two-dimensional NMR實驗分析HP0404與Zn2+的鍵結… 82 7.4 HP0404的蛋白質結晶…………………………………………82 第四章 結論（Conclusion）………………………… 83 參考文獻（References）………………………….… …86 圖表（Figures and Tables）………………….. ……….90

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