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論文中文名稱:利用線性轉殖與特定點重組製備戴奧辛之生物吸附劑 [以論文名稱查詢館藏系統]
論文英文名稱:Construction of cellular adsorbents for dioxin binding using linear transformation and site-specific recombination [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
中文姓名:林明樞
英文姓名:Ming-Shu Lin
研究生學號:93688007
學位類別:碩士
語文別:中文
口試日期:2006-07-28
論文頁數:87
指導教授中文名:侯劭毅
指導教授英文名:Shao-Yi Hou
口試委員中文名:黃志宏;王勝仕
口試委員英文名:Chih-Hung Huang;Sheng-Shih Wang
中文關鍵詞:線性轉殖同源序列特定點重組生物吸附戴奧辛
英文關鍵詞:linear transformationhomologous sequencesite-specific recombinationcellular adsorbentsdioxin
論文中文摘要:此研究論文主要的目的是利用線性DNA轉殖與特定點重組的方法,將能夠與戴奧辛結合的胺基酸表達在大腸桿菌的膜上,製備生物吸附劑。利用線性轉殖將線性DNA轉移到大腸桿菌的染色體上。如此一來便無須利用質體,且能表達重組基因的蛋白質。
這一段線性DNA,包含兩段FRT序列間的抗藥性基因,與目標核酸序列,核酸序列所轉譯出的胺基酸能夠與戴奧辛做一個穩定的結合,以λ red system做線性轉殖後,將質體pCP20轉型到剛剛重組成功的菌株,質體pCP20會表達重組蛋白酶FLP,FLP的作用能夠將兩段FRT之中的抗抗生素基因摘除。
為了確定這樣的方式是可以穩定的表達重組蛋白質,我們先以lacZ來測試線性轉殖的結果,實驗結果發現,若線性轉殖的位置附近有chi site的存在,可能會造成重組蛋白的不穩定表達,因此在進行線性重組時要避免chi site的存在。
這一套系統,只需要將目標核酸序列置換,便可以吸附不同的物質,且大量生產時,不需添加任何的抗生素即能使重組蛋白質穩定的表達,適合工業上的生產,並且能夠應用於自然環境之中。
論文英文摘要:The purpose of this study is to construct cellular adsorbents that could bind dioxin, using linear transformation and site-specific recombination. The feature of this method is to express recombinant proteins or peptides on E. coli outer membrane without plasmid and antibiotic selection.
The functional linear DNA fragment including the target sequence and the antibiotic resistance gene between two FLP recombination targets(FRT) sites was transformed into the chromosome of E. coli byλred system. After the transformants were cultured under antibiotic selection, the antibiotic resistance gene were eliminated by using a plasmid pCP20 expressing the flippase(FLP) recombinase, which acts on the directly repeated FRT sites flanking the antibiotic resistance gene.
In order to ensure the success of linear transformation, a DNA fragment, lacZ-FRT-cat-FRT, was transformed on two different sites of chromosome of E. coli. The result showed chi site has a connection with instable expression of recombinant protein. Hence, It is better that there is no chi site around the chosen integration location of linear transformation.
This system has a wide application for other compounds with binding peptide or protein. The system can continuously express recombinant protein without any antibiotic drug selection. It is preferred for industrial production and nature environment.
論文目次:中文摘要...................................i
英文摘要..................................ii
誌謝.....................................iii
目次......................................iv
圖目錄....................................vi
表目錄...................................vii

第一章 簡介.....................................1
1.1.戴奧辛污染..................................1
1.2.細胞表面蛋白質表現系統......................2
1.3.線性轉殖....................................3
1.4.特定點重組..................................3
1.5.本研究策略簡介..............................4
1.6.研究動機與目的..............................5
第二章 文獻探討.................................6
2.1.戴奧辛的檢測................................6
2.2.細胞表面蛋白質表現系統......................7
2.3.同源重組與λ Red系統.........................9
2.3.1.Exo,Beta與Gam............................11
2.4.特定點重組 FRT/FLP系統.....................14
2.5.本研究策略.................................15
第三章 實驗材料與方法..........................18
3.1.實驗方法...................................18
3.2.實驗儀器與設備.............................20
3.3.實驗藥品...................................22
3.4.實驗材料...................................25
3.4.1.菌種.....................................25
3.4.2.質體.....................................26
3.5.培養基.....................................26
3.6.重組質體構築...............................28
3.6.1.PCR 引子設計.............................28
3.6.2.聚合酶鏈鎖反應...........................29
3.6.3.瓊脂糖膠電泳.............................30
3.6.4.質體純化.................................32
3.6.5.限制酶酶切...............................33
3.6.6.連接酶接合反應...........................34
3.6.7.轉型作用.................................34
3.7.線性轉殖...................................36
3.7.1.同源序列 PCR 引子設計....................36
3.7.2.以 PCR 擴增線性 DNA 片段.................37
3.7.3.λ Red 線性轉殖...........................37
3.8.FLP/FRT 系統作用...........................38
第四章 實驗結果................................40
4.1.線性轉殖...................................40
4.1.1.選擇線性重組位置並以X-gal測試............40
4.2.pTrcFLDQV構築..............................47
4.2.1.pTrclpp’構築............................47
4.2.2.pTrcFLDQV構築............................52
4.3.pKD3FLDQV構築..............................58
4.4.線性轉殖構築菌株 VJS676FLDQV-cat...........65
4.5.FLP/FRT系統去除cat基因.....................70
4.6.PCR確認....................................72
第五章 結論....................................73
參考文獻.......................................75
附錄...........................................82
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