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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82171完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王勝仕(Sheng-Shih Wang) | |
| dc.contributor.author | Yi-Syue Guo | en |
| dc.contributor.author | 郭宜學 | zh_TW |
| dc.date.accessioned | 2022-11-25T06:33:09Z | - |
| dc.date.copyright | 2021-11-11 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-11 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82171 | - |
| dc.description.abstract | " 白內障全世界第三大宗的視力損傷疾病，起因為人類水晶體中的水晶體蛋白聚集。人類γ型水晶體蛋白為水晶體中的主要結構蛋白之一，被認為是白內障致病蛋白的主因之一，其中諸多γ類型的蛋白已研究多年，但人類γC型水晶體蛋白的相關研究相對較少，極具研究潛力。 人類γC型水晶體蛋白(Human γC Crystallin, HγCC)具有173個殘基，與人類γD型水晶體蛋白有71%的序列同一性，為富含β-sheet結構的結構域(domain)構成。本篇研究透過基因工程方式建立質體pEHisHγCC，轉入E.coli. BL21(DE3)系統進行表達，透過小量生產方式篩選出最適化的生產條件：30℃、0.5 mM IPTG、誘導前O.D.600 nm為1.5、誘導時間12小時，隨後以此條件進行大量生產，成功純化出帶有6×Histidine親和性標籤的HγCC，每100 ml培養液蛋白質產量為10.19±1.13 mg，並透過質譜分析與電泳分析確認分子量、純度達94.27%。 為了測定在不同條件下的HγCC聚集行為，我們透過濁度、ThT螢光光譜、ANS螢光光譜、Tryptophan螢光光譜、遠紫外光圓二色光譜與傅立葉轉換紅外線光譜等分析，證實pH 2酸性條件會誘導HγCC結構展開，錯誤摺疊而生成類澱粉纖維，整體二級結構比例變化趨勢為β-sheet下降、Random增加，再透過文獻模型擬和得到類澱粉纖維生長曲線，此外，也透過共軛聚焦顯微鏡與穿透式電子顯微鏡檢測聚集體的微觀型態，發現蛋白質會形成纖維網路結構，且55℃生成的結構相較37℃更為緊密；而在pH 7中性條件下，37℃培養後仍維持原態蛋白結構，而55℃培養後則會生成極少量的纖維網路，伴隨著些許的疏水區域裸露，但對整體三級結構與二級結構比例影響不大；最後，透過動態光散射法分析聚集物的尺寸分佈，發現酸性培養生成的聚集體尺寸遠大於中性培養生成的聚集體。 " | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T06:33:09Z (GMT). No. of bitstreams: 1 U0001-1008202113474800.pdf: 5188431 bytes, checksum: c41b52c6cd202cc85ab39a9e864914dc (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "目錄 誌謝 I 摘要 II Abstract III 目錄 V 圖目錄 VIII 表目錄 X 第一章 緒論與研究動機 1 第二章 文獻回顧 3 2-1人類水晶體(Lens) 3 2-1-1 水晶體分化 3 2-1-2 水晶體抗氧化機制 4 2-2白內障(Cataract) 6 2-2-1 白內障成因與類型 7 2-2-2 白內障治療 13 2-3水晶體蛋白(Crystallins) 14 2-3-1 人類α型水晶體蛋白 16 2-3-2 人類β/γ型水晶體蛋白 18 2-3-3 人類γC型水晶體蛋白 20 2-4 pH值對水晶體蛋白(Crystallins)的聚集行為影響 30 2-5 溫度對水晶體蛋白(Crystallins)的聚集行為影響 31 第三章 實驗儀器、藥品與步驟 33 3-1 實驗儀器 33 3-2 菌株 34 3-3 質體與純化套組 34 3-4 實驗藥品 36 3-5 溶液配製 37 3-6 實驗步驟 39 3-6-1 pEHisHγCC質體表現與純化 39 3-6-1-1 製作勝任細胞(Competent cell) 39 3-6-1-2 pEHisHγCC質體轉殖 40 3-6-1-3 pEHisHγCC質體純化 40 3-6-1-4 HγCC蛋白質生產─試管測試[66] 41 3-6-1-5 HγCC蛋白質生產─大量生產 43 3-6-1-6 定性分析(SDS-PAGE) 46 3-6-1-7 定量分析(BCA assay) 46 3-6-2 pH值、溫度誘導HγCC展開及聚集[63] 47 3-6-2-1 樣品配置 47 3-6-2-2 濁度(turbidity)量測 47 3-6-2-3 Tryptophan螢光光譜 47 3-6-2-4 Thioflavin T(ThT)螢光光譜 48 3-6-2-5 8-Anilino-1-naphthalenesulfonic acid(ANS)螢光光譜 48 3-6-2-6 圓二色(Circular Dichroism, CD)光譜量測 49 3-6-2-7 傅立葉轉換紅外線(Fourier-Transform Infrared, FTIR)光譜量測 49 3-6-2-8 動態光散射(Dynamic Light Scattering, DLS)量測 49 3-6-2-9 共軛聚焦顯微鏡(Confocal Microscopy)分析 50 3-6-2-10 穿透式電子顯微鏡(Transmission Electron Microscopy, TEM)分析 50 第四章 結果與討論 51 4-1 pEHisHγCC建立與表達 51 4-2 HγCC產量初步最適化─試管測試 53 4-2-1 試管活化測試 53 4-2-2 誘導條件測試 54 4-3 HγCC產量最適化─大量生產 61 4-3-1 活化測試 61 4-3-2 定性分析 63 4-3-3 定量分析 64 4-3-4 最佳誘導條件 68 4-4 pH值、溫度誘導HγCC展開及聚集 69 4-4-1 濁度(Turbidity)分析 72 4-4-2 Thioflavin T(ThT)螢光光譜分析 73 4-4-3 ANS螢光光譜分析 77 4-4-4 Tryptophan螢光光譜分析 81 4-4-5 遠紫外光圓二色(Far Ultraviolet Circular Dichroism)光譜分析 83 4-4-6 動態光散射(DLS)分析 86 4-4-8 共軛聚焦顯微鏡分析 88 第五章 結論 90 第六章 未來展望 92 第七章 參考文獻 93 附錄 99 附錄一 pET30b(+)圖譜 99 附錄二 合成後6×His-HγCC基因片段定序 99 附錄三 質體純化後6×His-HγCC基因片段定序 100 附錄四 HγCC的純度分析(SDS-PAGE) 101 附錄五 HγCC在pH 2、不同溫度下之傅立葉轉換紅外光譜(FTIR)圖 103 附錄六 HγCC在不同pH、溫度下之穿透式電子顯微鏡(TEM)圖 104 圖目錄 Figure 2-1 人體眼球與水晶體剖面圖[7] 3 Figure 2-2 人類水晶體分化過程[8] 4 Figure 2-3 穀胱甘肽(GSH)與過氧化物反應機制示意圖(以superoxide為例) 5 Figure 2-4 成人白內障發病病理模型[15] 6 Figure 2-5 核型白內障水晶體透明度分類系統III [16] 7 Figure 2-6 核硬化患者患部圖 8 Figure 2-7 隨年紀增加的水晶體色素沉澱切片圖 9 Figure 2-8 核型(B)、皮質型(C)、後囊型(D)白內障患部圖[17] 9 Figure 2-9 遺傳性白內障例子：(A)前囊下型(anterior subcapsular)(B)胎兒核型(fetal nuclear)(C)皮質點狀型(punctate in cortex)(D)胚胎Y核型(embryonic nuclear Y-sutural)[14]。 10 Figure 2-10 飛秒雷射白內障手術[25]。 14 Figure 2-11 水晶體中蛋白質間的互相作用(protein-protein interaction)示意圖[30] 16 Figure 2-12 希臘之鑰模組(Greek key motif)示意圖[6] 18 Figure 2-13 人類βB3型水晶體蛋白─trimer(PDB：3QK3) 19 Figure 2-14 人類γS型水晶體蛋白(PDB：2M3T) 19 Figure 2-15 人類γC型水晶體蛋白(PDB：2NBR) 20 Figure 2-16 人類γC型水晶體蛋白2D拓譜圖(topology) 20 Figure 2-17 人類γC型水晶體蛋白二級結構序列圖。 21 Figure 2-18 HγDC的tyrosine corners和與其作用的殘基[6]。 23 Figure 2-19 HγCC的WT與W157X結構圖[41]。 24 Figure 2-20 HγCC在R168附近的結構圖[42]。 24 Figure 2-21 HγCC在Y46附近的氫鍵網路圖[43]。 25 Figure 2-22 HγCC重區域置換示意圖[45] 25 Figure 2-23 類澱粉纖維(amyloid fibrils)形成路徑示意圖[62] 30 Figure 2-24 F(XNtd, XC-td, Xint)投影到[XNtd, XC-td, Xint at T=TNtd][45]。 32 Figure 3-1 pUC57(HγCC)質體圖譜 35 Figure 3-2 pEHisHγCC質體圖譜 35 Figure 4-1 pEHisHγCC質體建立示意圖 51 Figure 4-2 隨時間測試勝任細胞經pEHisHγCC轉殖後的培養菌落外觀 52 Figure 4-3 IPTG誘導目標基因表達示意圖 53 Figure 4-4 pEHisHγCC E.coli BL21(DE3)活化生長曲線(5 ml LB、37°C、164 rpm) 54 Figure 4-5 HγCC試管誘導電泳圖(0 ~ 9 hrs) 55 Figure 4-6 HγCC試管誘導電泳圖之一(0 ~ 15 hrs) 57 Figure 4-7 HγCC試管誘導電泳圖之二(0 ~ 15 hrs) 59 Figure 4-8 pEHisHγCC E.coli BL21(DE3)活化生長曲線(50 ml LB、37°C、164 rpm) 62 Figure 4-9 HγCC大量誘導電泳圖 63 Figure 4-10 HγCC大量誘導質譜圖。 64 Figure 4-11 BCA protein assay檢量線(BSA protein為standard)。 66 Figure 4-12 A280 protein assay檢量線(HγCC(不含β-Me)為standard)。 66 Figure 4-13 透析β-Me前後對tryptophan螢光光譜的影響。 67 Figure 4-14 HγCC在不同pH值與不同溫度下之濁度隨時間變化圖。 73 Figure 4-15 HγCC在pH 2、不同溫度下之ThT螢光強度隨時間柱狀圖。 74 Figure 4-16 HγCC在pH 2、55°C下之ThT螢光強度隨時間動力學fitting。 75 Figure 4-17 HγCC在pH 7、不同溫度下之ThT螢光強度隨時間柱狀圖。 77 Figure 4-18 HγCC在pH 2、不同溫度下的ANS螢光光譜隨時間變化圖 78 Figure 4-19 HγCC在pH 7、不同溫度下的ANS螢光光譜隨時間變化圖 80 Figure 4-20 HγCC在pH 2、不同溫度下的Tryptophan螢光光譜隨時間變化圖 82 Figure 4-21 HγCC在pH 7、不同溫度下的Tryptophan螢光光譜隨時間變化圖 83 Figure 4-22 HγCC在不同pH、溫度下之遠紫外光圓二色光譜圖 84 Figure 4-23 HγCC在pH 2、溫度下之二級結構比例柱狀圖。 85 Figure 4-24 HγCC在pH 7、溫度下之二級結構比例柱狀圖。 85 Figure 4-25 HγCC在不同pH、溫度下動態光散射法的尺寸分布圖 87 Figure 4-26 HγCC在不同pH、溫度下共軛聚焦顯微鏡圖 88 Figure 5-1 HγCC在不同pH、溫度下的聚集行為示意圖 91 表目錄 Table 2-1 代謝型白內障隨年紀分類[20] 11 Table 2-2 白內障風險因素整理[17] 12 Table 2-3 白內障保護因素整理[17] 12 Table 2-4 人類水晶體蛋白基本資訊[27-29] 15 Table 2-5 熱休克蛋白(HSPs)基本資訊[32] 17 Table 2-6 人類β/γ水晶體蛋白與HγCC的序列同一性 22 Table 2-7 人類γC型水晶體蛋白的近年研究統整 27 Table 4-1 HγCC試管誘導image J電泳分析圖(0 ~ 9 hrs) 56 Table 4-2 HγCC試管誘導image J電泳分析圖之一(0 ~ 15 hrs) 58 Table 4-3 HγCC試管誘導image J電泳分析圖之二(0 ~ 15 hrs) 60 Table 4-4 還原劑β-Me對BCA protein assay的影響 65 Table 4-5 透析前HγCC(含β-Me)與透析後HγCC(不含β-Me)的蛋白質濃度 67 Table 4-6 HγCC不同O.D600下大量誘導產量 68 Table 4-7 HγCC不同O.D600及不同鹽類濃度保存測試 68 Table 4-8 探討不同環境條件下的蛋白質聚集相關文獻 70 Table 4-9 類澱粉纖維生長曲線的近年文獻整理 75 " | |
| dc.language.iso | zh-TW | |
| dc.subject | 類澱粉纖維 | zh_TW |
| dc.subject | 蛋白質表達 | zh_TW |
| dc.subject | 人類γC型水晶體蛋白 | zh_TW |
| dc.subject | 白內障 | zh_TW |
| dc.subject | 溫度 | zh_TW |
| dc.subject | 蛋白質聚集 | zh_TW |
| dc.subject | 酸性 | zh_TW |
| dc.subject | amyloid fibril | en |
| dc.subject | Cataract | en |
| dc.subject | human γC crystallin | en |
| dc.subject | protein expression | en |
| dc.subject | low pH | en |
| dc.subject | temperature | en |
| dc.subject | protein aggregation | en |
| dc.title | 人類γC型水晶體蛋白聚集行為之研究 | zh_TW |
| dc.title | A Study of the Aggregation Behavior of Human γC Crystallin Proteins | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林達顯(Hsin-Tsai Liu),賴進此(Chih-Yang Tseng),侯劭毅,蔡伸隆,吳宛儒 | |
| dc.subject.keyword | 白內障,人類γC型水晶體蛋白,蛋白質表達,酸性,溫度,蛋白質聚集,類澱粉纖維, | zh_TW |
| dc.subject.keyword | Cataract,human γC crystallin,protein expression,low pH,temperature,protein aggregation,amyloid fibril, | en |
| dc.relation.page | 104 | |
| dc.identifier.doi | 10.6342/NTU202102244 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-08-11 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 化學工程學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-08-10 | - |
| 顯示於系所單位： | 化學工程學系 | |
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