臺灣博碩士論文加值系統

眼球中的水晶體，是脊椎動物身體中唯一透明的組織，負責將通過瞳孔的光線正確地聚焦於視網膜上進而轉換成大腦中的影像。水晶體蛋白是水晶體內主要的結構蛋白，讓水晶體能準確地折射光線。水晶體乾重有超過百分之九十以上是水晶體蛋白。在脊椎動物動物中，通常存在著三類泛存性水晶體蛋白，即 alpha-, beta-，及 gamma-水晶體蛋白。alpha-水晶體蛋白，同時也是小熱休克蛋白家族的成員之一，在體內扮演著重要的蛋白分子保護者的角色，預防因為熱，化學物質，和紫外線所引起的蛋白凝集現象產生。 在這篇論文中，我們利用了高解析度的二維膠體電泳以及基質輔助雷射脫附離子化-飛行時間-質譜儀分析豬和鯰魚的水晶體全蛋白的分布。並接續使用生物資訊學的資料庫進行序列的比對分析和月生 月太 斷片分子量比較分析以鑑定出正確的蛋白質身份。實驗結果顯示 alpha-, beta-，及 gamma-水晶體蛋白普遍存在於這兩種具有不同演化地位的物種的水晶體中。但是，兩物種間的水晶體蛋白在蛋白質的電荷組成及蛋白質分子量還是存有相當大的差異。

The lens of the eye, which is crucial for light focusing, is the only transparent tissue in the vertebrate body. Crystallins are the major structural proteins of the eye lens that are responsible for its light refractive power. The crystallins make up more than 90% of the total dry mass of the lens. In vertebrates, there are in general, three classes of ubiquitous crystallins, i.e. alpha-, beta-, and gamma-crystallins. alpha-Crystallin, a member of small heat shock protein family, plays an important role as a molecular chaperone to prevent thermo-, chemical-, and UV- induced protein aggregation. In this thesis, we analyzed the global protein profiles of pig and catfish lenses by using high-resolution two-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-Mass). Protein spots were identified by bioinformatics databases search and peptide mass comparison. The results revealed the common presence of alpha-, beta-, and gamma-crystallins in three evolutionarily distant species. Distinct differences in charge heterogeneity and molecular size of various crystallins can be found between these two species.

目錄
第一章 序論 1

第二章 實驗材料與方法 14

實驗材料 15

壹、 藥品試劑 15
貳、 主要儀器及器材 16
參、 試劑的配製 17

實驗方法 21

2.1水晶體樣品的處理與蛋白的萃取 21
2.2蛋白質定量 (by Bradford Method) 21
2.3水晶體蛋白質的純化 23
2.4蛋白質一維電泳 (SDS-PAGE) 24
2.5蛋白質二維電泳 (2-DE) 25
2.6 Coomassie Blue G-250膠片染色法 27
2.7 Pro-Q DIAMOND Phosphoprotein Gel Stain膠片染色法 28
2.8 SYPRO Ruby Gel Stain膠片染色法 29
2.9 In - Gel Digestion 30
2.10 ZipTip 純化 32

第三章 實驗結果 34

3.1 豬和鯰魚水晶體蛋白的純化 35
3.2 原態蛋白質分子量的鑑定 35
3.3 豬水晶體蛋白蛋白質次單元的鑑定 35
3.4 鯰魚水晶體蛋白蛋白質次單元的鑑定 36
3.5 蛋白保護子活性的測試 37
3.6 豬和鯰魚水晶體蛋白次單元的身份鑑定 37

第四章 結果討論 56

第五章 未來展望 60

第六章 參考文獻 62

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