臺灣博碩士論文加值系統

生物取像系統(bioimaging system)使用螢光蛋白當作報導蛋白，ㄧ般應用於真核生物，可即時精確地觀察到螢光蛋白在細胞或組織之形成及分布狀態；而原核生物細胞體積只有真核生物細胞的千分之ㄧ，通常無法使用生物取像系統精確地觀察螢光蛋白在胞內之分布狀態。本研究以特殊培養方法，將基因重組大腸桿菌BL21(DE3)/pET-D7做成與酵母菌同等大小之巨大原生質體(giant protoplast)。在IPTG 誘導下，由於藍色螢光蛋白-D7 的大量表現，可即時觀察到內涵體之形成，藉此可評估基因重組大腸桿菌形成內涵體之培養條件。
實驗發現在不同培養條件下，藉由螢光顯微鏡觀察巨大原生質體螢光表現差異，可得到隨著誘導劑及培養溫度的增加或培養液pH 值的下降，其內涵體的形成有增加的趨勢。

The bioimaging system usually utilizes fluorescence protein as a reporter gene in eukaryotic systems, and it gives us an easy way to real-time monitor the distribution of fluorescence protein in cells. Eukaryote is thousand times the size of prokaryote, so it is not suitable to this system. In our research, giant protoplasts with size similar to Saccharomycete were prepared from recombinant Escherichia coli BL21(DE3)/pET-D7. The expression of D7 can be induced by IPTG to monitor inclusion body formation in real time. Using this approach, we can study inclusion body formation in recombinant Escherichia coli under various culture conditions.
By the fluorescence microscope, we monitored the expression of fluorescence in the giant protoplast. The difference in fluorescence under various culture contitions revealed that inclusion body formation would
increase with increasing IPTG, with increasing induction temperature or with decreasing pH value.

中文摘要............................................................................................... I
英文摘要............................................................................................... II
誌謝....................................................................................................... III
目錄....................................................................................................... IV

表目錄................................................................................................... VIII
圖目錄................................................................................................... IV

符號表................................................................................................... Ⅹ

第一章 緒論......................................................................................... 1
1-1 前言................................................................................................ 1
1-2 細菌細胞外部構造簡介.................................................................. 2
1-2-1 細菌細胞外部構造............................................................... 2
1-2-2 革蘭氏陰性菌外部構造....................................................... 3
1-3 原生質體 (protoplast) 簡介.......................................................... 8
1-4 革蘭氏陰性菌原生質體形成方法.................................................. 9
1-5 生物冷光蛋白................................................................................ 12
1-5-1 創傷弧菌 (Vibrio vulnificus) 藍色螢光蛋白....................... 12
1-6 重組基因的誘導............................................................................ 16
1-7 內涵體 (inclusion body) 介紹....................................................... 18
1-8報導基因 (reporter gene) .............................................................. 19
1-9生物螢光影像................................................................................. 19
1-10研究動機與目的........................................................................... 20

第二章 實驗材料與方法...................................................................... 22
2-1 實驗材料........................................................................................ 22
2-1-1 藥品..................................................................................... 22
2-1-2 實驗儀器............................................................................. 24
2-1-3 菌株.................................................................................... 25
2-1-4 培養基、緩衝液與Stock Solution....................................... 27
2-2 實驗方法........................................................................................ 29
2-2-1 菌種保存與活化.................................................................. 29
2-2-2 原生質體之製備與培養....................................................... 30
2-2-3 不同培養條件下的內涵體製備............................................ 31
2-2-4 內涵體的純化................................................................. 32
2-3 分析方法................................................................................…. 34
2-3-1 原生質體圖像分析.......................................................... 34
2-3-2 內涵體活性分析.................................................................. 35
2-3-3 內涵體比例分析.................................................................. 35
2-3-4 原生質體成長與大小分析................................................... 36

第三章 結果與討論.............................................................................. 37
3-1 滲透壓對巨大原生質體的影響...................................................... 37
3-2 內涵體是否有活性......................................................................... 41
3-2-1 純化後D7之內涵體是否有活性......................................... 41
3-2-2 純化後的D7與VP1GFP融合蛋白之內涵體比較............ 43
3-3 內涵體的判定及比例分析............................................................. 48
3-3-1 內涵體的判定..................................................................... 48
3-3-2 內涵體的比例分析.............................................................. 49
3-4 不同培養條件下之內涵體比例...................................................... 51
3-4-1 誘導溫度對內涵體生成的影響........................................... 51
3-4-2 誘導劑最終濃度對內涵體生成的影響................................ 53
3-4-3 培養液pH值對內涵體生成的影響..................................... 55

第四章 結論......................................................................................... 58
未來展望............................................................................................... 60
參考文獻............................................................................................... 61
自述....................................................................................................... 65

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