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研究生:江英鍵
研究生(外文):Ying-Jian Jiang
論文名稱:發展細菌之可程式細胞瓦解系統以回收蛋白質
論文名稱(外文):Development of the programmed bacterial cell lysis system for protein recovery
指導教授:趙雲鵬
指導教授(外文):Yun-Peng Chao
口試日期:2010-01-05
學位類別:碩士
校院名稱:逢甲大學
系所名稱:生醫資訊暨生醫工程碩士學位學程
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:64
中文關鍵詞:噬菌體高溫誘導
外文關鍵詞:phageheat induction
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  本研究是將噬菌體基因選殖於以溫度調控之啟動子的質體,當含此質體的細菌,藉由高溫的誘導後,以使得噬菌體蛋白產生而破裂細胞。
  此外,我們將溫度調控之啟動子做定點突變,以增加其嚴密調控性。實驗結果顯示,此舉可對菌株DH5α有較好之成效,然對菌株BL21 (DE3)之效果較差。基於此,我們又透過基因工程改質,以提升破菌效果。最後,將改良後之系統運用於大腸桿菌中,以利胞內生產之重組蛋白質的後續回收。
In this study, the phage gene of interest was subcloned and placed under the control of the thermo-inducible promoter on the plasmid. Upon thermal induction, the host strain harboring this plasmid is expected to express the phage gene product which would cause the autolysis of cells.
In addition, the heat-responsive promoter was mutated by site-directed mutagenesis to increase its stringent regulation. As a result of experiments, it was found that the system functioned superiorly on Escherichia coli strain DH5α over strain BL21 (DE3). Moreover, the system was engineered to improve its functionality. Finally with the application of this modified system, the recombinant protein produced in E. coli strain was released extracellularly, in a hope to facilitate the downstream processing of the protein.
目 錄
第一章 緒論
1.1 前言………………………………………………………… 1
1.2 文獻回顧…………………………………………………… 2
1.3 研究動機及目的…………………………………………… 3
第二章 實驗材料與方法
2.1 實驗器材………………………………………………………… 5
2.2 菌種之儲存與馴養……………………………………………… 6
2.2.1 菌種儲存………………………………………………… 6
2.2.2 菌種馴養………………………………………………… 6
2.2.3 培養基的配製…………………………………………… 6
2.3 DNA質體純化…………………………………………………… 7
2.4 勝任細胞的準備………………………………………………… 8
2.5 轉殖作用………………………………………………………… 9
2.6 聚合酵素連鎖反應……………………………………………… 9
2.6.1 大量複製所欲之特定基因因………………………………… 9
2.6.2 定點突變……………………………………………………… 10
2.7 轉導作用………………………………………………………… 12
2.7.1 P1vir 溶胞產物製備………………………………………… 12
2.7.2以P1vir溶胞產物進行轉導作用………………………… 14
2.8 質體建構與菌種製備…………………………………………… 15
2.8.1建構質體pPLφXE……………………………………………… 15
2.8.2建構質體pPL*φXE…………………………………………… 15
2.8.3建構質體pPLφXE2…………………………………………… 15
2.8.4建構質體pPLSφXE2…………………………………………… 16
2.8.5建構質體pPLSφX2E…………………………………………… 16
2.8.6建構質體pφ80-X2E…………………………………………… 16
2.8.7建構質體pPL-LacZ…………………………………………… 17
2.8.8建構質體pPL*-LacZ…………………………………………… 17
2.8.9建構質體pPLS-LacZ…………………………………………… 17
2.8.10建構菌種BL21(X2E)…………………………………………… 17
2.8.11建構菌種EN(X2E)…………………………………………… 17
第三章 結果與討論………………………………………………… 33
第四章 結論與未來展望…………………………………………… 60
參考文獻 …………………………………………………………… 62
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