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研究生： 林鼎翔
Ting-Shiang Lin
論文名稱： 胃螺旋幽門桿菌蛋白質HP0404結構與功能的探討
Solution-structural and functional studies of protein HP0404 from helicobacter pylori
指導教授： 程家維
Jay-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： 2006
畢業學年度： 94
語文別： 中文
論文頁數： 81
中文關鍵詞： 核磁共振光譜儀 、螺旋幽門桿菌
外文關鍵詞： NMR, Helicobacter pylori
相關次數： 點閱：1  下載：0 
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    • 胃螺旋幽門桿菌 (Helicobacter pylori)是一種格蘭氏陰性菌、成長慢、具有鞭毛及螺旋外狀的細菌，他會引起嚴重的胃潰瘍、十二指腸相關的疾病和胃癌。根據蛋白質序列比對的結果，由於一段特殊的片段片段 (motif)：His-X-His-X-His-X-X (其中X是疏水性的胺基酸)我們發現HP0404蛋白質是一種屬於HIT (histidine triad)蛋白質家族裡的蛋白質激酶的抑制劑 (protein kinase C inhibitor)。HIT 蛋白質家族主要分為三個分支：Fhit (fragile histidine triad)分支，這一類主要是在動物和真菌中被發現。HINT (histidine triad nucleotide-binding protein)分支，這一類的蛋白質幾乎出現在所有的細胞中，另外最後一個是Galt (Galactose-1-phosphate uridylytransferase)分支。在這篇論文中，我們選擇HP0404作為我們的研究目標，利用NMR (nuclear magnetic resonance)來研究其結構， 利用C13 和N15標定的HP0404蛋白質樣品做NMR的實驗，並且也辨認出來HP0404的氫、碳、氮的化學位移的數值，光譜的分析的部分利用將同核二維的核磁共振光譜搭配了C13和 N15的三維核磁共振光譜(實驗都在298K PH=6.5的環境下進行)，在這裡，我們呈現HP0404蛋白質的序列分析的結果，並且將分析的結果標示在HSQC (heteronuclear single quantum correlation)光譜圖上。並且也做了腺甘三磷酸(adenosine 5′-triphospate)滴定的實驗，發現I17、N20、S25、S44、K68、T81、N86 和H97這些胺基酸在蛋白質和ATP作用中也許扮演著重要的角色。希望得到的結果可以對之後HP0404蛋白質結構和功能研究有更進一步的幫助。

    H. Pylori (Helicobacter pylori) is a Gram-negative, microaerophilic, and slow-growing bacterium, which can cause serious gastric ulcer, duodenum-associated disease and gastric cancer. According to the sequence alignment, HP0404 which is produced by H. pylori is the PKC inhibitor of HIT (histidine triad) protein family, because of the special sequence motif, His-X-His-X-His-X-X (X is a hydrophobic amino acid). HIT proteins fall into three branches, the Fhit (fragile histidine triad) branch that is found only in animals and fungi and histidine triad nucleotide-binding protein (Hint) branch that has represented in all cellular life and the Galt(Galactose-1-phosphate uridylytransferase) brench. Here we chose HP0404 as interest target to investigate the structure by using NMR spectroscopy (nuclear magnetic resonance). The C13- and N15-label protein sample of HP0404 have been produced for the NMR experiment. Proton, nitrogen, and carbon chemical shift assignment have been made for HP0404. assignment were made from a combination of homonuclear two-dimensional and N15- and C13- edited three –dimensional spectra at PH6.5 and 298K. Here we present the sequential assignment of HP0404 and make the residue annotation on the HSQC (heteronuclear single quantum correlation) spectra. We also did the ATP titration experiment for functional study and found residue I17, N20, S25, S44, K68, T81, N86 and H97 maybe important for interacting with ATP. This annotation maybe could offer slight help for further investigation between structure and function of HP0404.

    第一章 序論 7 一、結構基因體學 (STRUCTURAL GENOMICS) 7 二、幽門螺旋桿菌 (HELICOBACTER PYLORI) 7 三、HP0404蛋白質的目標基因篩選 10 四、PKCI (PROTEIN KINASE C INHIBITOR )蛋白質的功能及結構研究 11 五、HP0404與PKC INHIBITOR的關係 14 六、HP0404蛋白質的研究目標 14 第二章 材料與方法 16 一、HP0404 CLONING 16 1.1基因選殖 16 1.2 載體 (VECTOR)：PETBLUE-2 16 1.3 引子 (Primers)合成 17 1.4 聚合酶鏈鎖反應 (PCR，Polymerase chain reaction) 18 1.5 瓊酯凝膠電泳 (Agarose gel electrophoresis) 18 1.6 限制酵素反應 (Restriction enzyme reaction) 19 1.7 接合作用 (Ligation) 20 1.8.1 勝任細胞之製備 (Competent cell preparation) 20 1.8.2 勝任細胞之檢測 21 1.9 重組質體之轉形作用 (Transformation) 22 二、HP0404蛋白質表現 22 2.1 蛋白質大量表現 (large scale expression) 22 2.2 蛋白質純化 (protein purification) 24 2.3 SDS-PAGE分析 (SDS-PAGE analysis) 25 三、ATP TITRATION EXPERIMENT 25 3.1 樣品的配製 25 四、核磁共振（NMR） 25 4.1樣品製備 25 3.2 核磁共振的基本參數 26 3.3 核磁共振實驗 27 4.4 核磁共振光譜處理與分析 (Data processing and analysis) 34 第三章 實驗結果與討論 35 一、HP0404基因篩選 35 二、HP0404蛋白質的表現和純化 35 三、蛋白質結構分析 36 3.1 One dimensional NMR spectroscopy 36 3.2 Two dimensional NMR spectroscopy 36 3.3 Three dimensional NMR spectroscopy 37 3.4 NMR實驗結果分析 39 3.5 二級結構的預測 40 3.6 ATP titration experiment 42 第四章 未來發展 44 圖表 圖一、HELICOBACTER PYLORI的電子顯微鏡圖（A）與UREASE（B）作用示意圖 48 圖二、幽門螺旋桿菌HELICOBACTER PYLORI週遭的PH分布圖 49 圖三、幽門螺旋桿菌(HELICOBACTER PYLORI)進入胃中的感染路徑引起疾病的主要因子。（NATURE REVIEW , 2001, 2, 457-466） 50 圖四、HELICOBACTER PYLORI 26695染色體基因圓形示意圖 51 TABLE-1 52 HP0404在TIGR DATABASE之基因相關資訊 52 圖五、HP0404 DNA SEQUENCE AND PROTEIN SEQUENCE 53 圖六、純化出的蛋白質HP0404 WILD TYPE胺基酸序列 53 圖七、HP0404 的 SDS-PAGE 54 圖八、利用選擇性標示標示得到的HP0404蛋白質的SDS電泳圖。 55 圖九、ONE DIMENSIONAL SPECTRUM OF HP0404 56 圖十、HP0404蛋白質的HSQC (HETERO-NUCLEAR SIGNAL QUANTUM CORRELATION)光圖譜(298 K PH 6.5) 57 圖十一、HP0404 利用HNCA (黑色)和HNCOCA (紅色)的連線示意圖 58 圖十二、HP0404的CBCACONH (A)和HNCO (B)的光譜圖 59 圖十三、HSQC SPECTRUM OF ILE SELECTIVE-LABEL HP0404 60 圖十四、ILE（綠色）選擇性標定和一般HSQC的疊圖 61 圖十五、HSQC SPECTRUM OF LEU SELECTIVE-LABEL HP0404 62 圖十六、LEU（黃色）選擇性標定和一般HSQC的疊圖 63 圖十七、HSQC SPECTRUM OF LYS SELECTIVE-LABEL HP0404 64 圖十八、LYS（藍色）選擇性標定和一般HSQC的疊圖 65 圖十九、HSQC SPECTRUM OF TYR SELECTIVE-LABEL HP0404 66 圖二十、TYR（粉紅色）選擇性標定和一般HSQC的疊圖 67 圖二十一、HSQC SPECTRUM OF VAL SELECTIVE-LABEL HP0404 68 圖二十二、VAL（淡藍色）選擇性標定和一般HSQC的疊圖 69 圖二十三、HSQC SPECTRUM OF ALA SELECTIVE-LABEL HP0404 70 圖二十四、ALA（紅色）選擇性標定和一般HSQC的疊圖 71 圖二十五、六種選擇性標示（SELECTIVE-LABEL）和HSQC的疊圖（ILE IN GREEN, LEU IN YELLOW, LYS IN BLUE, ALA IN RED, VAL IN CYAN, TYR IN PINK） 72 圖二十六、HP0404序列分析 (SEQUCIAL ASSIGNMENT )的HSQC圖譜 73 圖二十七、HP0404 CSI (CHEMICAL SHIFT INDEX)二級結構預測結果 74 圖二十八、HINT (HISTIDINE TRIAD NUCLEOTIDE BINDING PROTEIN ) HUMAN 77 蛋白質的二級結構分析圖 77 圖二十九、不同濃度的HP0404抑制PKC使用ATP 磷酸化 78 圖三十、ATP TITRATION 的HSQC圖 79 圖三十一、HP0404 在ATP和蛋白質濃度比五比一和沒有加入ATP 80 的情況下的化學位移變化(CHEMICAL SHIFT CHANGE)的示意圖 80 註：訊號消失的部分，定義為變動一個百萬分之一 (PPM)。下面藍色的箭頭圖形 80 代表利用CSI計算得來HP0404的二級結構的預測圖。 80

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