臺灣博碩士論文加值系統

氧化鯊烯環化酵素（OSC）在固醇類和三萜類產物的生合成中，扮演重要的角色，可以催化受質鯊烯或者氧化鯊烯進行環化/重組反應，轉變成多環結構的羊毛硬脂醇及環阿屯醇。由於氧化鯊烯環化酵素催化的複雜產物膽固醇及麥角脂醇是構成細胞膜的重要要素，且在細胞生長過程中產生此酵素，所以氧化鯊烯環化酵素作為目標物設計抗菌劑及降低膽固醇藥物是相當重要的課題。我們對於篩選氧化鯊烯環化酵素有親合力作用的胜肽序列感到興趣，希望未來發展成為藥物。用兩種噬菌體胜肽庫，七個及十二個隨機胜肽的胜肽庫，對氧化鯊烯環化酵素進行配位基的篩選。將噬菌體胜肽庫和覆蓋滿目標物的盤子反應，洗去為鍵結的噬菌體，並且將有鍵結的噬菌體洗滌下來，依照這樣的過程進行五次對氧化鯊烯環化酵素的親和篩選，得到具有鍵結力的噬菌體胜肽序列，並且進行DNA定序，得到八個對氧化鯊烯環化酵素具有親和性的噬菌體胜肽序列，由西方墨點法測定得到1個七胜肽序列和6個十二胜肽序列具有反應。結果中，其中1個表現十二胜肽序列的樣品在點墨法中清楚看到反應，但在ELISA測試中反應較弱，利用西方墨點法測試時，卻沒有反應。

目錄 頁次
中文摘要………………………………………………...……Ⅰ
英文摘要…………………………………………...………....Ⅱ
目錄…………………………………………………...………Ⅲ
圖目錄……………………………………………………...…Ⅵ
表目錄…………………………………………………...……Ⅷ

第一章 緒論

1-1 膽固醇的重要性…………………………………………...………1
1-2 膽固醇生合成…………………………………………...…………3
1-3 氧化鯊烯環化酵素之簡介……………………………………...…5
1-3-1 氧化鯊烯環化酵素的酵素學…………………………...…….7
1-3-2 氧化鯊烯環化酵素的反應機制……………………...……….9
1-4 噬菌體表現系統之簡介…………………………………………..15
1-5 噬菌體表現系統的型式…………………………………………..18
1-6 噬菌體胜肽庫(peptide library)及抗體庫(antibody library)…20
1-7 噬菌體表現系統篩選方式………………………………………...21
1-8 研究動機與目的…………………………………………………...22

第二章 實驗材料及方法

實驗材料
2-1 藥品………………………………………………………………..24
2-2緩衝液及溶液配置………………………………………...………27
2-3 實驗儀器…………………………………………………………..31
2-4 菌株及質體………………………………………………………..32
實驗方法
2-5 牛肝中氧化鯊烯環酵素之純化…………………………………..33
2-5-1 溶解微粒體…………………………………………...……...34
2-5-2 粗萃取液……………………………………………………..34
2-5-3 Q-Sepharose陰離子交換管柱層析………………………….34
2-5-4 Hydroxyapatite Gel管柱層析………………………………..35
2-5-5 HiTrap Heparin 管柱層析…………………………………...35
2-5-6 酵素的分子量及純度分析………………………………….35
2-5-7 酵素活性測試……………………………………………….37
2-5-8 酵素濃度分析……………………………………………….38
2-6 Ph.D.-C7C及Ph.D.-12對牛肝鯊烯環化酵素的親和篩選……..39
2-6-1 牛肝鯊烯環化酵素之製備………………………………….40
2-6-2負向親和篩選………………………………………………..41
2-6-3 第一次親和篩選…………………………………………….41
2-6-4 將噬菌體培養放大………………………….……………….41
2-6-5 第二次親和篩選…………………………………………….42
2-7 噬菌體效價之測定………………………………………………43
2-7-1 方法一：培養盤菌落數計………………………………….43
2-7-2 方法二：光譜吸收測定…….……………………………….43
2-8噬菌體胜肽庫對氧化鯊烯環化酵素之親和力測定……….44
2-8-1 酵素連結免疫吸附分析法 (ELISA)之反應測試……….44
2-8-2 墨點法 (Dot blot)之反應測試……………………………...45
2-8-3 西方墨點法 (Western blotting)之反應測試………………..45
2-9 單股DNA模版之純化…………………………………………..47
2-10 DNA定序………………………………………………………..47
2-11 單株抗體的製備………………………………………………...49
2-11-1 免疫動物……………….……………….…………………...49
2-11-2 骨髓癌細胞 (myeloma cell) …………………….……...49
2-11-3 細胞融合……………………………………………………51
2-11-4 細胞增殖及取樣篩檢………………………………………51
2-11-5 融合細胞之單株化………….………………………………52
2-11-6 單株抗體之產生………….…………………………………52
2-12利用酵母菌系統表現牛肝氧化鯊烯環化酵素…………………53
2-11-1 聚合酶連鎖反應…………………………………….……..54
2-11-2 質體的建構………………………………………………...55
2-11-3 功能性互補…….…………………………………………...55
2-11-4 酵母菌株TKW14C2的質體轉化作用…………………...56
2-11-5 質體交換………………………………………...…………...56
2-11-6 酵母菌株pichia strain (GS115)的質體轉化作用………58
2-11-7聚合酶連鎖反應分析重組DNA…………………………..58
2-11-8 利用酵母菌系統表現蛋白質………...…………………...59

第三章 實驗結果與討論

3-1 氧化鯊烯環化酵素之純化………………………….………….…60
3-2 氧化鯊烯環化酵素的單株抗體…………………….…………….61
3-3 噬菌體胜肽庫親合篩選…………………….……………………62
3-3-1 噬菌體效價測定…………………………….…………….67
3-3-2 利用墨點法作親和力測試………………………..………67
3-3-3 利用酵素連結免疫吸附分析法作親和力測試…..………68
3-3-4 利用西方墨點法作親和力測試………………….………….69
3-3-5 噬菌體胜肽庫之定序…………………….….…………….72
3-4 利用酵母菌系統表現牛肝中氧化鯊烯環化酵素…………….74

第四章 結論與未來展望……………………………………..……75

第五章 參考資料………………………………………...……….…76

附錄…………………………………………………………………….79
附錄一 Pichia pastoris表現牛肝氧化鯊烯環化酵素…………..,……79



圖目錄

圖1-1 膽固醇的化學結構…………………………………………..…1
圖1-2 膽固醇在動物體內的代謝途徑………………………………..4
圖1-3 動物體內氧化鯊烯環化酵素的催化反應…………………..5
圖1-4 自然界各物種間的(氧化)鯊烯環化酵素作用途徑….………6
圖1-5 (氧化)鯊烯環化酵素產生的相異結構產物……………………9
圖1-6 (氧化)鯊烯環化酵素環化反應機制及產物多樣性…………...11
圖1-7 Johnson Model示意圖…………...……….………………….12
圖1-8 Q-W motif理論的模型示意圖…….………...………………13
圖1-9 噬菌體感染大腸桿菌的生命週期…………………………….15
圖1-10 絲狀噬菌體pⅢ感染大腸桿菌過程…………………………16
圖1-11 絲狀噬菌體結構示意圖……………………………………...17
圖1-12 噬菌體表現外來蛋白質的兩種型式………………………...18
圖1-13 SIP技術示意圖……………….………….………………….19
圖1-14 在ELISA盤子上篩選過程的示意圖………………..……..21
圖2-1 TLC活性測試示意圖………………………………………..37
圖2-2 噬菌體胜肽庫篩選示意圖…………………………………..40
圖2-3 噬菌體光譜吸收圖…………………………………………..43
圖2-4 抗原抗體測定示意圖………………………………………..44
圖2-5 西方墨點法三明治疊法……………………………………..46
圖2-6 PCR定序條件………………………………………………48
圖2-7 骨髓癌細胞正規合成路徑及旁支輔助路徑……………….50
圖2-8 酵母菌中DNA重組………………………………………..54
圖2-9 PCR反應條件………………………………………………..54
圖2-10 功能性互補示意圖………………………………………..…57
圖3-1 牛肝氧化鯊烯環化酵素SDS-PAGE…………………………60
圖3-2 西方墨點法測定單株抗體對氧化鯊烯環化酵素之專一性…61
圖3-3 七個隨機胺基酸序列胜肽庫對氧化鯊烯環化酵素第二次及第三次親和篩選ELISA鍵結力測試結果………………………………63
圖3-4 (A)噬菌體Ph.D.-C7C胜肽庫和氧化鯊烯環化酵素ELISA反應結果 (B)噬菌體Ph.D.-12胜肽庫和氧化鯊烯環化酵素ELISA反應結果………………………………………………………………………65
圖3-5 墨點法測定單一噬菌體對氧化鯊烯環化酵素親和性結果…67
圖3-6 ELISA方法測定單一噬菌體對氧化鯊烯環化酵素親和性結果………………………………………………………………………68
圖3-7 西方墨點法測定單一噬菌體對氧化鯊烯環化酵素的親和性結果………………………………………………………………………70
圖3-8 功能性互補的篩選結果示意圖……………………………74






表目錄

表1-1 自然界中不同物種氧化鯊烯環化酵素的純化效果及特性……7
表1-2 絲狀噬菌體的基因、蛋白質及功能介紹………………………17
表2-1 牛肝中氧化鯊烯環化酵素的純化流程簡圖…………………..33
表2-2 12 ％及4 ％蛋白質電泳膠片配方……………………………36
表2-3 rTth polymerase PCR反應試劑的量…………………………...55
表2-4 Taq polymerase PCR反應試劑的量……………………………60
表3-1 噬菌體七個胜肽庫對氧化鯊烯環化酵素第三次親和篩選得到的序列…………………………………………………………………..63
表3-2 噬菌體胜肽庫對氧化鯊烯環化酵素進行篩選得到的序列…..72

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