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研究生:許翼軒
研究生(外文):Hsu, Yi-Hsuan
論文名稱:Thermus thermophilus HB8核苷酸激酶與核苷二磷酸激酶基因之選殖與應用於去氧核醣核苷三磷酸之合成
論文名稱(外文):Cloning of Deoxynucleoside Monophosphate Kinase and Deoxynucleoside Diphosphate Kinase Genes from Thermus thermophilus HB8 and Establishment of a New Enzymatic Method for Biosynthesis of Deoxynucleoside Triphosphates
指導教授:張晃猷
指導教授(外文):Chang, Hwan-You
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:63
中文關鍵詞:嗜熱菌HB8核苷酸激酶核苷二磷酸激酶去氧核醣核苷三磷酸
外文關鍵詞:Thermus thermophilus HB8Deoxynucleoside Monophosphate KinaseDeoxynucleoside Diphosphate KinaseDeoxynucleoside Triphosphate
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去氧核醣核苷三磷酸 (dNTP) 是生物體合成去氧核醣核酸必要的原料,也是分子生物學技術如聚合酶鏈鎖反應中常用的反應物。由於生物體dNTP含量不高,不易萃取,因此市面上販售的dNTP主要以化學合成方式製造,但通常產率不高且須使用毒性溶劑。本研究目的為建立在體外以酵素合成dNTP的系統,以解決這些問題。此生產過程以去氧核醣核苷單磷酸為起始反應物,先產生去氧核醣核苷二磷酸再進而生成dNTP。我們從嗜熱菌Thermus thermophilus HB8中選殖四個核苷酸激酶與一個核苷二磷酸激酶,分別在大腸桿菌中大量表現重組蛋白質。隨後並對此些酵素進行功能性與動力學特性分析。結果顯示此五種酵素在pH 7.0到pH 8.0且溫度介於75度至80度時表現出最佳活性。此外,在75度環境下處理三小時都可保有80%以上活性,即使處理24小時之後仍能保有50%以上活性,顯示這五種酵素具有極佳的熱穩定性。在動力學試驗測試中也發現此五種酵素在其最佳反應環境下都表現出較嗜溫性菌還高的催化效率。我們組合這些酵素進行初步dNTP生產,並將所得到的產物用於聚合酶鏈鎖反應進行檢測,結果顯示與市面上販售的dNTP無明顯差異。此一系統將有潛力應用於工業上,以乾淨有效的方式生產dNTP。
Deoxynucleoside triphosphates (dNTP) are essential biological materials for synthesizing DNA. The compounds are also used in molecular biotechnology such as the polymerase chain reaction (PCR) and other DNA polymerase-based applications. Nowadays, dNTPs are commercially produced by chemical synthesis due to their low concentrations in living organisms. However, the chemical synthesis method utilizes toxic solvents and the yield is low. To solve these problems, this study attempt to establish an in vitro enzymatic method for complete biosynthesis of dNTP. The process involves two stages of enzymatic phosphorylation procedures. The deoxynucleoside monophosphates (dNMPs) are used in the first step. The products of the first and second stages are deoxynucleoside diphosphates (dNDP) and dNTP, respectively. Five genes from the Thermus thermophilus HB8, which encode four kinds of deoxynucleoside monophosphate kinases (NMKs) and one nucleoside diphosphate kinase (NDK), were cloned and expressed in high quality in E. coli BL21 (DE3) and NovaBlue (DE3). We have performed functional and kinetic property analysis of NMKs and NDK. The results showed that the five enzymes exhibited optimum activity at the pH value of 7.0 to 8.0 and at the temperature between 75OC to 80 OC. Furthermore, all of them displayed excellent thermal stability. Approximate 80% and 50% of enzyme activities were retained after incubation at 75 OC for 3 hr and 24hr, respectively. The kinetic property analysis showed that five enzymes have higher catalytic efficiency under optimum environment as compared to the mesophilic bacteria. Later, we combined these crude enzymes preliminarily to produce dNTP. The PCR performed using the enzymes-synthesized dNTP showed the same efficiency compared to the commercial one. Finally, this system provides a clean and better method of preparation that has potential for commercialization.
中文摘要………………………………………………………………………………I
英文摘要…………………………………………………………………………….. II
致謝…………………………………………………………………………………..III
縮寫字對照表……………………………………………………………………..…IV
目錄…………………………………………………………………………………VII
表目錄………………………………………………………………………………..IX
圖目錄………………………………………………………………………………...X
壹、 前言………………………………………………………………………………1
貳、 材料與實驗方法…………………………………………………………………8
2.1. 菌株與生長環境…………………………………………………………....8
2.2. 基因體DNA萃取………………………………………………………..…8
2.3. 聚合酶鏈鎖反應……………………………………………………………9
2.4. 接合作用…………………………………………………………………....9
2.5. 勝任細胞製備………………………………………………………………9
2.6. 熱休克轉型法……………………………………………………………..10
2.7. 小量質體DNA純化………………………………………...………….....10
2.8. 質體構築…………………………………………………….……...……..11
2.9. 蛋白質表現並利用His6-tag親和性管柱純化………...…….……...……12
2.10. 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳……………………….….............13
2.11. 酵素最佳活性測定- pH的影響...………………………………....……...14
2.12. 酵素最佳活性測定-反應溫度的影響…………………………………….15
2.13. 酵素熱穩定性測試………………………………………………………..15
2.14. 酵素動力學特性分析………………………………………....…………..16
2.15. 純化之NMKs與NDK合成dNTP……………………………..……….17
2.16. 以高效液相層析儀進行dNTP生合成之定性分析……………………...17
參、 結果…………………………………………………………………....………..19
3.1. 分析T. thermophilus HB8的NMKs與NDK之胺基酸特性分………...19
3.2. 構築表現載體與重組蛋白質之表現純化………………………………..20
3.3. NMKs重組蛋白質之最適反應環境分析……………………...………...21
3.4. NMKs重組蛋白質之熱穩定性測試…………………………...………...22
3.5. NMKs重組蛋白質之酵素動力學特性分析…………………………......23
3.6. 建立新穎的dNTP生產系統進行dNTP生產與使用………………..….24
3.7. 利用高效液相層析儀進行dNTP之生合成之定性及定量分析………...24
肆、 討論……………………………………………………………………………..26
伍、 參考文獻…………………………………………………………………….….33

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