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研究生:邱亭錡
研究生(外文):Ting-Ci Ciou
論文名稱:利用重組技術對土番鴨水晶體 epsilon 晶體蛋白進行基因表現、鑑定與拉曼光譜結構分析
論文名稱(外文):Gene Cloning, Expression and Raman Spectroscopic Characterization of epsilon-Crystallin from Mule Duck Lens
指導教授:陳文龍陳文龍引用關係吳游源
指導教授(外文):Wenlung ChenYu-Yuan Wo
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:應用化學系研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
畢業學年度:96
語文別:中文
中文關鍵詞:傅立葉轉換拉曼光譜水晶體epsilon 晶體蛋白
外文關鍵詞:FT-Ramanlensepsilon-crystallin
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□ epsilon 晶體蛋白是部份鳥類及鱷魚水晶體中,除了 delta 晶體蛋白外另一主要的物種特異性晶體蛋白 (taxon-specific crystallin),且其含量約佔整個水晶體蛋白質的 23%。Wistow 等人證實來自於鴨子 (Anas platyrhynchos) 之 epsilon 晶體蛋白除了做為水晶體結構外,亦具有乳酸去氫&;#37238; (lactate dehydrogenase, LDH, EC 1.1.1.27) 的活性。而 Hendriks 等人進一步發現 epsilon 晶體蛋白之胺基酸序列與乳酸去氫&;#37238; (LDH-B4) 一致。回顧相關文獻,至今仍無土番鴨 epsilon 晶體蛋白之報導。
鴨子水晶體中的 epsilon 晶體蛋白,由於與其心肌中的乳酸去氫&;#37238; (LDH-B4) 在序列上有一致性,且仍保有其酵素活性,在過去已有許多相關研究發表。本論文研究乃利用基因轉殖技術及蛋白質純化技術探討野生型的土番鴨 epsilon 晶體蛋白之活性,並與從土番鴨眼睛組織中直接分離純化所得的 epsiln 晶體蛋白作比較。此外配合拉曼光譜技術直接偵測 epsilon 晶體蛋白結構。
結果顯示得到的土番鴨 epsilon 晶體蛋白在胺基酸序列 115 與 119 位置有 NF 與 GG 兩種基因型態,有異於北京鴨 (GG type) 及正番鴨 (NF type),此胺基酸序列上的改變可能是因為遺傳關係或者是選擇性壓力 (selective pressure),而造就了不同物種間上序列的差異。
在蛋白質結構方面,經由拉曼光譜偵測並配合 curve fitting 分析得知,由基因轉殖得到的土番鴨 epsilon 晶體蛋白之主要二級結構,與市售乳酸去氫&;#37238; (LDH-B) 或直接組織純化之 epsilon 晶體蛋白結構相一致:分別為 50% alpha-helix、35% anti-parallel beta-sheet 與 15% random coil。然而基因轉殖得到的 epsilon 晶體蛋白中,在雙硫鍵構形皆存在著 gauche-gauche-trans、trans-gauche-trans 與 gauche-gauche-gauche 三種形態,而分離純化的 epsilon 晶體蛋白卻以 gauche-gauche-trans、trans-gauche-trans 為主。另分析三者之酵素活性,發現其大小依序為:市售乳酸去氫&;#37238; (LDH-B)>分離純化 epsilon 晶體蛋>基因轉殖 epsilon 晶體蛋白。除了純化過程中可能造成活性差異外,真核生物體中的後修飾作用或者是胺基酸序列的改變可能也都是影響活性的因素之一。

The lenses of many birds and crocodiles contain in addition to alpha-, beta-, and delta-crystallins another major protein, epsilon-crystallin, which accounts for up to 23% of the total lens protein. Wistow et al. have showed that in addition to being a structural protein, epsilon-crystallin, is an active fermentation enzyme of lactate dehydrogenase (LDH, EC 1.1.1.27). Hendriks et al. indicated that duck lens epsilon-crystallin and duck heart LDH-B4 are the product of the same gene. Although epsilon-crystallin has been studied previously, there remain no reference related to the epsilon-crystallin of mule duck which is a hybrid species of Kaiya and Muscovy duck. In this thesis, mule duck epsilon-crystallin was first expressed by gene cloning and purified by immobilized metal-ion affinity chromatography (IMAC). The structure of cloned epsilon-crystallin was then studied by FT-Raman spectroscopy of which result was compared to the one isolated from lens tissue and commercially available LDH-B Then compared cloned one’s enzymatic activity with the one isolated from eye lenses and the commercially available LDH-B.
The secondary structure of LDH-B and epsilon-crystallins from lens tissue authentic and recombinant sources are in 50% alpha-helix, 35% beta-sheet, and 15% random coil. This result indicated that epsilon-crystallin, like delta-crystallin, but in contrast to alpha- and beta-crystallin, had a relatively high amount of alpha-helical structure. The S-S conformation of epsilon-crystallin (cloned) was different from that of tissue-isolated. For LDH activity assay, they also exhibited differently with an order: LDH-B > epsilon-crystallin (authentic) > epsilon-crystallin (cloned). The loss of LDH activity might be due to the process of purification, or the effect of post-translational modifications, which rarely occurred in bacteria. Some amino acid changed might also cause the alteration of protein stability. An inherent factor or selective pressure may contribute to the two gene types of epsilon-crystallin’s 115/119 amino residues in the mule duck, G/G and N/F types. Information about the protein structure was obtained by FT-Raman coupled with curve fitting.

中文摘要 …………………………………………………………… i
英文摘要 …………………………………………………………… iii
目錄 ………………………………………………………………… v
圖目錄 ………………………………………………………………viii
表目錄 ……………………………………………………………… x
第一章 緒論 ………………………………………………………… 1
1-1 眼睛與水晶體 ………………………………………………… 1
1-2 晶體蛋白 (Crystallin) ……………………………………… 3
1-2-1 普遍性晶體蛋白 (Ubiquitous crystallins) …………… 4
1-2-2 物種特異性晶體蛋白 (Taxon-specific crystallins) … 5
1-2-3 epsilon 晶體蛋白 (epsilon-crystallin) ……………… 7
1-3 鳥類的簡介 …………………………………………………… 8
1-4 乳酸去氫酶 …………………………………………………… 10
1-5 研究動機 ……………………………………………………… 12
第二章 拉曼光譜原理及應用 ……………………………………… 13
2-1 前言 …………………………………………………………… 13
2-2 散射原理 ……………………………………………………… 15
2-3 拉曼散射 ……………………………………………………… 15
2-3-1 古典理論 …………………………………………………… 16
2-3-2 量子理論 …………………………………………………… 19
2-4 振動光譜 ……………………………………………………… 23
2-5 拉曼光譜儀器設備 …………………………………………… 25
2-5-1 光源 ………………………………………………………… 25
2-5-2 分光儀 ……………………………………………………… 26
2-5-3 樣品照射系統 ……………………………………………… 27
2-5-4 偵測器 ……………………………………………………… 27
2-6 傅立葉轉換拉曼光譜 ………………………………………… 28
2-7 生化方面應用 ………………………………………………… 31
2-8 拉曼光譜對於鑑定蛋白質結構參考訊息 …………………… 32
第三章 材料與方法 ………………………………………………… 36
3-1 前言 …………………………………………………………… 36
3-2 儀器藥品 (附錄 A、B) ……………………………………… 36
3-3 實驗方法與步驟 ……………………………………………… 36
3-3-1 cDNA 的製備 ………………………………………………… 36
3-3-1-1 動物組織中萃取 RNA …………………………………… 36
3-3-1-2 RNA 轉錄成 cDNA ………………………………………… 38
3-3-2 引子設計 …………………………………………………… 38
3-3-3 聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) … 39
3-3-3-1 PCR 條件 ………………………………………………… 40
3-3-3-2 PCR 反應設定 …………………………………………… 40
3-3-4 PCI 萃取與酒精沉澱 ……………………………………… 43
3-3-5 限制性酵素剪切 …………………………………………… 44
3-3-6 挖膠純化 (Gel purification by electroelution) …… 44
3-3-7 PCR 產物黏接至 pET-21b vector (Ligation) ………… 45
3-3-8 預先篩選 (Preselection) ………………………………… 46
3-3-9 轉型至 E. coli DH5a ……………………………………… 48
3-3-9-1 勝任細胞 (competent cell) 的製備 ………………… 48
3-3-9-2 轉型作用 ………………………………………………… 49
3-3-10 快速少量抽取質體 DNA (Easypreps) …………………… 49
3-3-11 限制性酵素切割分析 ……………………………………… 50
3-3-12 DNA 定序 …………………………………………………… 50
3-3-13 鹼溶裂法抽取質體 DNA (Alkaline lysis) …………… 51
3-3-14 轉型至E. coli BL21 (DE3) ……………………………… 53
3-3-14-1 勝任細胞 (competent cell) 的製備 ………………… 53
3-3-14-2 轉型作用 ………………………………………………… 53
3-3-15 利用 IPTG 篩選菌落 ……………………………………… 54
3-3-16 蛋白質小量表現 …………………………………………… 55
3-3-17 蛋白質大量表現 …………………………………………… 56
3-3-18 蛋白質純化 (Protein purification)…………………… 57
3-3-18-1 利用 IMAC 來純化重組蛋白 …………………………… 57
3-3-18-2 去鹽 ……………………………………………………… 58
3-3-18-3 透析與冷凍乾燥 ………………………………………… 59
3-3-19 蛋白質定量 ………………………………………………… 59
3-3-19-1 蛋白質標準曲線的製作 ………………………………… 60
3-3-20 蛋白質電泳分析 (SDS-PAGE) …………………………… 61
3-3-21 epsilon 晶體蛋白酵素活性測定 ………………………… 63
3-3-21-1 酵素動力學 ……………………………………………… 65
3-3-22 拉曼光譜分析 ……………………………………………… 67
3-3-23 數據統計處理 ……………………………………………… 68
第四章 結果與討論 ………………………………………………… 69
4-1 前言 …………………………………………………………… 69
4-2 利用基因轉殖得到 epsilon 晶體蛋白與純化結果 ………… 71
4-3 epsilon 晶體蛋白胺基酸序列 115 與 119 位置之探討…… 80
4-4 epsilon 晶體蛋白之拉曼光譜結構分析 …………………… 85
4-5 epsilon 晶體蛋白/乳酸去氫酶酵素活性之探討 …………… 103
第五章 總結論 ……………………………………………………… 110
參考文獻 …………………………………………………………… 114
附錄 A ……………………………………………………………… 124
附錄 B ……………………………………………………………… 126
附錄 C ……………………………………………………………… 129
附錄 D ……………………………………………………………… 137
個人發表著作 ……………………………………………………… 138

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