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研究生： 卓淑鶯
Toh, Shu Ing
論文名稱： 人類DISC1和幽門螺旋桿菌AlpA粘附素之表達、純化與結晶
Expression, Purification and Crystallization of human DISC1 and AlpA from Helicobacter pylori
指導教授： 鄭惠春
Cheng, Hui Chun
口試委員: 孫玉珠
Sun, Yuh Ju
高茂傑
Kao, Mou Chieh
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年： 2016
畢業學年度： 104
語文別： 中文
論文頁數： 89
中文關鍵詞： 精神分裂症 、蛋白質 、結構 、功能 、胃幽門螺旋桿菌 、外膜蛋白 、純化 、結晶
外文關鍵詞： Schizophrenia, protein, structure, function, Helicobacter pylori, adhesion protein, purification, crystallizatoin
相關次數： 點閱：3  下載：0 
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    • 精神分裂症是一種破壞性的精神障礙，被認為已經影響了1 %的全世界人口。過去研究發現DISC1基因是一種精神分裂症的分子指標，所以DISC1基因可以用來評估精神分裂疾病。但DISC1蛋白質的結構與功能之間的關係尚未被瞭解。瞭解DISC1較有功能性的結構區域，對於相關的科學應用會更有幫助。
    另外，我們研究造成胃炎、胃潰瘍與胃癌等胃疾病的胃幽門螺旋桿菌外膜蛋白AlpA。AlpA的基因可以產生有助於胃幽門螺旋桿菌附著在胃上皮的蛋白。我們希望可以更瞭解AlpA蛋白質結構與功能之間的關係，這樣將會對治療與預防胃幽門螺旋桿菌所引起之胃疾病有很大的突破。
    我們利用大腸桿菌表現DISC1和AlpA蛋白。再利用各種純化蛋白方法，如：Ni2+離子親和性管柱、分子篩層析、離子交換管柱、直鏈澱粉親和管柱等方法來純化DISC1_S1、S2及S3和AlpA-N2各個蛋白片段。利用蛋白結晶試劑，如：Hampton PEG/Ion HTTM、 PCT kit及Qiagen JCSG Screen等方法尋找適合DISC1及AlpA蛋白長出晶體的蛋白質濃度及蛋白結晶試劑條件。
    本研究成功建立了這兩種蛋白質的純化方法。未來希望可以找尋適合這兩種蛋白長出晶體的蛋白質濃度及結晶試劑的條件，大量長出晶體，利用Ｘ光單晶繞射學解析出蛋白質晶體結構，運用於藥物設計及進一步功能上的研究。

    Schizophrenia is a devastating mental disorder and is believed to have affected 1 % of the world's population. Previous research found that the DISC1 gene is a kind of the molecular marker of spirit split disorder, but the relationship between the structure and function of the DISC1 protein is not understood. An atomic model of DISC1 will help us better understand the function of DISC1.
    Helicobacter pylori cause gastritis, gastric ulcer and gastric cancer. The AlpA protein is a H. pylori adhesion protein that attaches to the host gastric epithelium. We hope to better understand the relationship between the structure and function of AlpA protein, which may be beneficial for the treatment and preventatoin of Helicobacter pylori-mediated stomach diseases.
    We used the E.coli system to overexpress DISC1 and AlpA proteins and used various protein purification methods, such as: Ni2+ affinity column chromatography, gel filtration chromatography, ion exchange chromatography, and amylose affinity chromatography to purify these proteins. We used protein crystallization screens, such as Hampton PEG/Ion HTTM, PCT kit and Qiagen JCSG screens to find suitable conditions for DISC1 and AlpA crystallizatoin.
    This study successfully established the purification methods for various constructs of DISC1 and AlpA. However, we did not get any crystallization hits after several trials. Hopefully, our work may facilitate the crystallization experiments and further functional studies in the future.

    目　　錄 摘　　要 I Abstract II 致 謝 III 目　　錄 IV 表　目　錄 VI 圖　目　錄 VII 第一章、Disrupted-in-Schizophrenia-1（DISC1） 1 第一節、簡介 1 與DISC1相關的疾病 1 DISC1在細胞生物學上的影響 2 DISC1的蛋白質結構研究 3 對DISC1的研究動機 5 第二節、研究程序 6 克隆 6 表達蛋白 6 純化程序 7 結晶篩選 12 第三節、研究結果 15 通過PCR分析DISC1基因分子克隆 15 純化各個DISC1片段蛋白 15 結晶篩選 18 第四節、討論 Disrupted-in-Schizophrenia-1(DISC1) 21 Cloning DISC1 constructs 22 Purification of DISC1 constructs 23 DISC1蛋白的結晶篩選 27 第二章、Adherence associated lipoproteins A（AlpA） 28 第一節、簡介 28 幽門螺旋桿菌Helicobacter pylori（H.pylori）與胃疾病 28 幽門螺旋桿菌Helicobacter pylori（H.pylori）相關的外膜蛋白 28 AlpA的預測結構與功能 29 AlpA在細胞生物學上的研究 30 對AlpA的研究動機 30 第二節、研究程序 32 克隆 32 表達蛋白 32 純化程序 32 結晶篩選 34 第三節、研究結果 35 通過PCR分析AlpA-N2基因分子克隆 35 純化AlpA-N2蛋白 35 結晶篩選 36 第四節、討論Adherence associated lipoproteins A(AlpA) 37 Purification constructs of AlpA-N2 38 Crystallization Screen of AlpA-N2 problem 38 第三章、研究材料 39 第四章、參考文獻 42 附錄 53 一、全長的DISC1基因及氨基酸序列 53 二、AlpA全長的基因與氨基酸序列 57 表　目　錄 表1‑1. PCR condition for DISC1 59 表1‑2. Primers’ sequences 60 表1‑3. Reaction condition for Gibson ligation 61 表1‑4. The extinction coefficient and molecular weight of proteins 62 表1‑5. PCT results 63 表2‑1. Reaction conditions for AlpA-N2 PCR 64 圖　目　錄 圖1-1. DISC1 interacting proteins and associations with human mental disorders 65 圖1-2. Classification of DISC1 network 66 圖1-3. p15Cool2-MBP-tev vector 67 圖1-4. 離子親和性管柱層析系統設置的流程梯度圖 68 圖1-5. Crsytal hit 69 圖1-6. Crystallization screen of DISC1_S1 70 圖1-7. Results of DISC1_S2 and DISC1_S3 PCT 71 圖1-8. DISC1二級結構的預測圖 72 圖1-9. PCR results of DISC1_S2 and DISC1_S3 73 圖1-10. 表達、純化DISC1_S1蛋白的結果 74 圖1-11. 表達、純化DISC1_S2蛋白的結果 75 圖1-12. 表達、純化DISC1_S3蛋白的結果 76 圖1-13. DISC1二級結構的概述 79 圖2-1. Electron micrograph of H. pylori 80 圖2-2. Type V secretion pathway for autotransporters 81 圖2-3. p15Cool2 vector 82 圖2-4. AlpA二級結構的預測圖 83 圖2-5. 表達、純化AlpA-N2蛋白的結果 84 圖2-6. 表達、重新折疊AlpA-N1蛋白的結果 86 圖2-7. 純化AlpA-N1蛋白的結果 87 圖2-8. 設計AlpA-N2胞外結構域的示意圖 88 圖2-9. 以MS分析AlpA-N2蛋白樣品 89

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