臺灣博碩士論文加值系統

摘 要
本項論文的研究目標為應用已知p53蛋白質驗證創新技術平臺，結合酵母菌單雜合系統 (Yeast One-Hybrid System) 與循環式目標擴增選拔 (Cyclic Amplification and Selection of Targets, CASTing) 的交互實驗應用，取其安全有效優點獲得特定p53蛋白質專一性結合基因序列，運用p53轉錄因子活化區與DNA結合區專一鍵結特性，獲得對側轉錄因子p53蛋白質的調控基因序列與共識結合序列 (Consensus binding seequence)，改進傳統方法無法同時檢測多個未知目標與實驗耗時之缺點，提供需時較短、專一性高，多個目標進行同時檢測的創新技術平臺
本項研究以對側轉錄因子 (Trans) 酵母菌表現菌株，重組循環式目標擴增選拔 (Cyclic Amplification and Selection of Targets, CASTing) 核酸模板的報導基因載體系統，建立創新應用單雜合系統篩選驗證集存庫的CASTing核酸結合序列，經序列排比獲致該對側蛋白質之共識同側結合序列。應用單雜合系統篩選p53對側轉錄因子酵母菌表現株相關CASTing核酸，確證CASTing核酸模板區別性與同側序列 (Cis-) 調控性，利用p53-CASTing核酸及pLacZi質體建立單雜合系統β-gal報導載體之同側序列集存庫，轉形p53對側轉錄因子酵母菌表現菌株，進行X-gal藍白篩選系統確認選殖CSATing核酸序列。選取對側p53轉錄因子表現之藍色菌落，定序獲得對側轉錄因子調控序列，進行序列比對獲得p53蛋白質結合共識序列，達成建立驗證本項技術平臺。
關鍵字：調控基因序列、共識結合序列、p53蛋白質、循環式目標擴增選拔、酵母菌單雜合系統、藍白篩選系統

Summary
The goal in research of this thesis, in order to use known p53 protein to prove that innovate the technological platform, combine the saccharomycete form and mix the system of shutting (Yeast One-Hybrid System) increase and choose with the circulation type goal (Cyclic Amplification and Selection of Targets, the mutual experiment application that CASTing ), fetch its safe effective advantage and obtain specific p53 protein uniqueness and combine the gene order, use the transcription factor activating area of p53 to form the characteristic with the single-minded key of combining area of DNA, get and combine the array (Consensus binding seequence ) with the common understanding to the regulation and control gene order of the side transcription factor p53 protein, it is unable to measure the shortcomings of a lot of unknown goals and experiments consuming time at the same time to improve the traditional method, it is higher than the short , uniqueness while offering taking, the technological platform of the innovation that a lot of goals measure at the same time
This research is by displaying the bacterial strain to side transcription factor (Trans ) and saccharomycete, recombinate the circulation type goal to increase and choose (Cyclic Amplification and Selection of Targets, CASTing), nucleic acid template report gene carrier system, is it is it is it is it shut system is it is it collect CASTing nucleic acid to check the storehouse combine arrays to prove to screen to mix only to use to innovate to set up, obtained the array by the parallelism of the array and should combine with side to the common understanding of the side protein. Use and mix and equal to the system and screen p53 and display a relevant CASTing nucleic acid to side transcription factor and saccharomycete only, prove conclusively CASTing nucleic acid template person who distinguish and with side array person who adjust and control, utilize p53-CASTing nucleic acid and pLacZi quality body is it is it shut systematic | Â to mix only to set up - gal report to is it check the storehouse to collect with side array carrier, transfer to shape p53 and display to side transcription factor and saccharomycete the bacterial strain, carry on X-gal and screen the system bluly and whitly and confirm being selected the bone CSATing nucleic acid array. Choosing the blue colony displayed to the side p53 transcription factor, the preface got and adjusted and controlled arrays to the side transcription factor definitely, carrying on arrays will combine the common understanding array than to obtaining p53 protein , reach and set up and verify this technological platform.
Key word: Adjust and control the gene order, common understanding combine the array , p53 protein , circulation type goal and increase and choose , the saccharomycete form mixes and shuts the system , screens the system bluly and whitly

目 錄
頁次
誌謝 …………………………………………………………………… I
中文摘要 ………………………………………………………………… II
英文摘要 ………………………………………………………………… III
目錄 …………………………………………………………………… IV
圖目錄 …………………………………………………………………… VIII
表目錄 …………………………………………………………………… X

第一章 緒論 ………………………………………………………… 1
第一節 研究背景 …………………………………………………… 1
第二節 研究目的 …………………………………………………… 2
第三節 章節摘要 …………………………………………………… 5

第二章 材料準備: 對側轉錄因子菌落及驗證 ………………… 7
第一節 文獻探討 ………………………………………………… 7
第二節 實驗材料 ………………………………………………… 9
一、對側轉錄因子質體 (Plasmid) …………………………… 9
二、酵母菌選擇型菌株YM4271 Yeast Strain ……………… 10
三、培養基及培養液配製 ……………………………………… 10
四、質體小量製備 ………………………………………………… 12
五、質體大量製備試劑組QUIAGEN Plasmid Maxi kit …… 13
六、酵母菌細胞核萃取物製備 ………………………………… 13
七、配製聚丙烯醯胺SDS-PAGE凝膠電泳溶液 …………… 14
八、寡核苷酸3端DIG標識 ………………………………… 15
九、免疫呈色試劑組 Western Breeze (Invitrogen) ………… 17
第三節 實驗方法 ………………………………………………… 18
一、對側轉錄因子質體大量製備 …………………………… 18
(一) 大腸桿菌勝任細胞製備 …………………………… 18
(二) 質體轉形至大腸桿菌 ……………………………… 18
(三) 質體大量製備 ……………………………………… 18
二、對側轉錄因子酵母菌株 ……………………………… 19
(一) 酵母菌勝任細胞製備 …………………………… 19
(二) 質體轉形酵母菌株 …………………………… 19
(三) 大腸桿菌小量質體備製 …………………………… 20
(四) 驗證質體 …………………………………………… 21
第四節 實驗結果 ………………………………………………… 24
一、轉形大腸桿菌質體驗證 …………………………………… 24
二、對側轉錄因子菌落挑選 …………………………………… 26
三、驗證對側轉錄因子質體 …………………………………… 27
(一) 電泳分析 ……………………………………………… 27
(二) 酵母菌蛋白質分析 …………………………………… 28
第五節 討論 ……………………………………………………… 32

第三章 循環式目標擴增選拔 …………………………………… 34
第一節 文獻探討 ………………………………………………… 34
一、循環式目標擴增選拔技術及應用 ……………………… 34
二、p53蛋白質 ………………………………………………… 35
第二節 實驗材料 ………………………………………………… 37
一、蛋白質結合隨機核酸模板 (Templates) ………………… 37
二、特定結合蛋白質 …………………………………………… 38
三、10X CBS CASTing Buffer ……………………………… 38
四、寡核苷酸3端DIG標識 ……………………………… 39
五、免疫沉降 Immuno-precipitation ………………………… 40
六、驗證CASTing技術平台 ……………………………… 41
(一) 人類互補核酸大腸桿菌表現集存庫
ProQuest cDNA Library …………… 41
(二) 酵母菌選擇型菌株MaV203 Yeast …………… 42
(三) pPC86質體擴增引子 ……………………………… 43
第三節 實驗方法 ………………………………………………… 43
一、循環式目標擴增選拔 …………………………………… 43
(一) 結合蛋白質 ………………………………………… 43
(二) 核酸模版擴增的製備 …………………………… 44
(三) 循環式目標擴增選拔CASTing ………………… 45
(四) 免疫沉降 ……………………………………………… 47
(五) 驗證CASTing實驗可行性 ………………………… 48
第四節 實驗結果 …………………………………………………… 55
一、核酸擴增模版的前期聚合酶鏈反應 (Pre-PCR) …… 55
二、Pre-PCR雙股核酸產物濃度 …………………………… 56
三、免疫沉降 …………………………………………………… 56
四、驗證CASTing實驗設計 …………………………… 58
(一) DIG標識效率 DIG Labeling Efficiency ……… 58
(二) 點陣列雜合反應Dot Array Hybridization ……… 59
(三) 驗證菌株質體DNA片段 …………………………… 62
第五節 討論 …………………………………………………… 65


第四章 酵母菌單雜合系統
…………………………………………
67
第一節 文獻探討 ………………………………………………… 67
一、酵母菌雜合系統 (Yeast “n” Hybrid System) ………… 67
第二節 實驗材料 ………………………………………………… 70
一、報導載體pLacZi …………………………………………… 70
二、質體大量製備 ……………………………………………… 71
三、酵母菌轉形作用 …………………………………………… 71
四、藍白篩選 ………………………………………………… 72
第三節 實驗方法 ………………………………………………… 72
一、pLacZi載體大量備製 …………………………………… 72
二、建構單雜合系統 β-gal 報導載體之同側序列集存庫 … 72
三、勝任細胞備製 (Preparation of competent cell) ………… 73
四、重組質體轉形至勝任細胞 …………………………… 73
五、X-gal藍白篩選系統 ……………………………………… 74
六、同側調控基因定序及定義共識序列 ………………… 75
第四節 實驗結果 ………………………………………………… 75
一、pLacZi質體大量備製定量 …………………………… 75
二、限制酶酵素Eco R I作用之載體電泳分析 ……… 76
三、藍白篩選 …………………………………………………… 77
第五節 討論 ………………………………………………………… 78

第五章 結論與未來展望 …………………………………………… 80

參考文獻 ………………………………………………………………… 82

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