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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 王愛玉(Ai-Yu Wang) | |
dc.contributor.author | Chih-Ying Chen | en |
dc.contributor.author | 陳芝瑩 | zh_TW |
dc.date.accessioned | 2021-06-08T04:37:47Z | - |
dc.date.copyright | 2009-08-19 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23013 | - |
dc.description.abstract | 普遍存在於生物體中的 Cytochrome P450 monooxygenases (CYP450s) 是一種血基質蛋白質 (hemoprotein),由一群大家族 (superfamily) 基因所表現。CYP450 參與許多的催化反應,包括 carbon hydroxylation、heteroatom oxygenation、dealkylation、epoxidation、aromatic hydroxylation、reduction 和 dehalogenation 等反應。市面上已有許多產品是應用 CYP450s的特性所開發,例如抗生素。
本論文利用不同的 PCR 方法,由 Nonomuraea angiospora 中選殖出 CYP450 基因。此菌株已被發現具有將 compactin 轉化成 pravastatin 的能力。Pravastatin為 HMG-CoA reductase 活性抑制因子,被用來降低血液中的膽固醇含量。Nonomuraea angiospora CYP450 基因的編碼區域 (open reading frame) 全長為 1224 bp,可轉譯成 407 個胺基酸,預估分子量為 45 kDa。Nonomuraea angiospora CYP450 之胺基酸序列與 Nonomuraea recticatena、Salinispora arenicola CNS-205 以及Saccharopolyspora erythraea NRRL2338 的 CYP450 分別具有 90%、66% 和 61 % 的同質性。由序列分析、二級結構預測及三級結構模擬分析結果,推測 Nonomuraea angiospora CYP450 隸屬於 CYP105 family,可以使用 mitochondrial/bacterial type 的電子傳遞系統,催化 compactin 轉化為 pravastatin 的反應。 | zh_TW |
dc.description.abstract | Cytochrome P450 monooxygenases (CYP450s), which are ubiquitously distributed in organism, are hemoproteins encoded by a superfamily of genes. They catalyze a variety of chemical reactions including carbon hydroxylation, heteroatom oxygenation, dealkylation, epoxidation, aromatic hydroxylation, reduction and dehalogenation. The enzyme family has been used for production of a lot commercial products such as antibiotics.
In this study, CYP450 gene was cloned from Nonomuraea angiospora, which has been found to be capable of converting compactin to pravastatin, by different PCR methods. Pravastatin is an inhibitor of HMG-CoA reductase and is used in reducing the cholesterol levels in blood. The open reading frame of Nonomuraea angiospora CYP450 gene consists of 1224 bp and is predicted to encode 407 amino acids with a molecular mass of 45 kDa. The amino acid sequence of Nonomuraea angiospora CYP450 shows 90%, 66% and 61% identities with CYP450s from Nonomuraea recticatena, Salinispora arenicola and Saccharopolyspora erythraea, respectively. The results of sequence analysis、secondary structure prediction and 3D structure modeling suggest that Nonomuraea angiospora CYP450 belongs to the CYP105 family and may catalyze the hydroxylation of compactin to pravastatin by using the mitochondrial/bacterial type electronic transport system. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T04:37:47Z (GMT). No. of bitstreams: 1 ntu-98-R96b47218-1.pdf: 2958362 bytes, checksum: ea2a317125040aa624b68611c2321435 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 目錄 I
摘要 IV ABSTRACT V 縮寫表 VI 第一章 緒論 1 1 膽固醇 (CHOLESTEROL) 1 1.1 膽固醇生理功能 1 1.2 膽固醇來源與生合成 2 1.3 膽固醇相關疾病 4 2 降膽固醇藥物發展史 4 2.1 1950s~1970s: 4 2.2 1970s 後: Statin 類藥物 6 2.2.1 Compactin 7 2.2.2 Lovastatin 8 2.2.3 Simvastatin 9 2.2.4 Pravastatin 9 2.2.5 其他 statin 類藥物 12 3 CYTOCHROME P450 MONOOXYGENASE 13 3.1 Cytochrome P450 monooxygenase 簡介 13 3.2 Cytochrome P450 monooxygenase 的分類 14 3.2.1 依命名分類 14 3.2.2 依據電子傳遞系統 (electron supporting system) 分類 15 3.3 Cytochrome P450 monooxygenase 反應機制 16 3.4 Cytochrome P450 monooxygenase 結構分析 17 3.5 Cytochrome P450 monooxygenase 的應用 19 3.5.1 藥物合成 (Drug synthesis) 19 3.5.2 生物整治 (Bioremediation) 20 4 放線菌 (ACTINOBACTERIA) 20 5 研究動機與目的 21 第二章 材料與方法 23 1 實驗菌種 23 1.1 放線菌 23 1.2 大腸桿菌 23 1.3 酵母菌 23 2 載體 24 3 實驗藥品 24 3.1 一般化學試劑: 24 3.2 分生實驗用藥品與限制酶酵素: 24 3.3 培養基: 24 4 實驗儀器 24 4.1 離心機 24 4.2 核酸電泳系統 25 4.3 蛋白質電泳系統 25 4.4 其他儀器 25 5 實驗方法 26 5.1 放線菌培養 26 5.1.1 固態及液態培養 26 5.1.2 冷凍孢子之製作 27 5.2 DNA 之純化與分析 27 5.2.1 放線菌基因組 DNA 之抽取 27 5.2.2 質體 DNA 之小量分離法 28 5.2.3 質體 DNA 之中量分離法 28 5.2.4 質體快速檢定法 29 5.2.5 DNA 瓊脂糖膠體電泳分析 29 5.2.6 DNA 片段之純化 30 5.2.7 DIG 探針製備 30 5.2.7.1 PCR製備 DNA 探針 30 5.2.7.2 定量探針 DNA 原液量 31 5.2.8 南方墨點法 (Southern blotting) 32 5.2.8.1 轉印 32 5.2.8.2 雜合反應 32 5.2.9 DIG 酵素連結免疫反應與與呈色反應 33 5.2.10 一般 PCR 反應 33 5.2.11 Inverse PCR 34 5.2.12 Tail-PCR 36 5.2.13 PCR 產物之 A-Tailing 反應 38 5.2.14 接合反應 39 5.2.15 大腸桿菌勝任細胞之製備 39 5.2.16 大腸桿菌之轉形 (Transformation) 40 5.2.17 大腸桿菌轉型株之篩選檢定 40 5.2.17.1 表現質體 40 5.2.17.2 pGEM T質體 40 5.2.18 菌種保存 41 第三章 結果與討論 42 1 ENV 1-8 及 ENV 2-1 之型態與生長曲線 42 1.1 菌絲形態 42 1.2 生長曲線 42 2 ENV 1-8 的 CYTOCHROME P450 MONOOXYGENASE 基因選殖 43 2.1 不同菌種 Cytochrome P450 monooxygenase 序列比對與 PCR 引子對設計 43 2.2 以 PCR 擴增 ENV1-8 及 ENV 2-1 之 CYP450 部分基因 44 2.3 以 Inverse PCR 選殖 ENV 1-8 CYP450 5 端及 3 端之序列 45 2.4 Anchored PCR 47 2.5 Tail-PCR 47 3 ENV 1-8 序列分析 48 3.1 ENV 1-8 CYP450 DNA 序列分析 48 3.2 ENV 1-8 CYP450 功能性區塊 (functional domain) 分析 49 3.3 一級結構分析 50 3.4 二級結構分析 50 3.5 三級結構預測 51 4 ENV 1-8 CYP450 重組蛋白質表現 52 4.1 表現載體 pPICZ B-CYP450 及 pYES2-CYP450 之建構 52 第四章 結論與未來展望 53 1 結論 53 2 未來展望 53 2.1 CYP450 蛋白質表現與分析 53 2.2 建構 CYP450 完整異體表現系統 (heterologous expression system) 54 2.3 蛋白質遺傳工程 (Protein engineering) 54 REFERENCES 55 圖與表 62 | |
dc.language.iso | zh-TW | |
dc.title | 放線菌 Nonomuraea angiospora 中 Cytochrome P450 Monooxygenase 之選殖 | zh_TW |
dc.title | Molecular Cloning of Cytochrome P450 Monooxygenase Gene from Nonomuraea angiospora | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 宋賢一,張珍田,林忠亮,楊健志 | |
dc.subject.keyword | pravastatin,compactin,HMG-CoA reductase,cytochrome P450 monooxygenase (CYP450),Nonomuraea angiospora, | zh_TW |
dc.relation.page | 89 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2009-08-17 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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