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論文中文名稱:重組蛋白質過度表達下的細胞溝通及質體不穩定現象 [以論文名稱查詢館藏系統]
論文英文名稱:Evidences for Cell Communication and Plasmid Instability Mechanism during Protein Overexpression in Escherichia coli. [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:化學工程所
中文姓名:陳建良
英文姓名:Chien-Liang Chen
研究生學號:92328002
學位類別:碩士
語文別:中文
口試日期:2005-07-11
論文頁數:62
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:顏宏偉;王勝仕
口試委員英文名:Hong-Wei Yen;Sheng-Shih Wang
中文關鍵詞:重組質體蛋白過量表達細胞溝通
英文關鍵詞:Recombinant plasmidProtein overexpressionCell communication
論文中文摘要:本篇論文為探討以質體(plasmid)為載體(vector)時,在蛋白質過度表達(protein overexpression)下大腸桿菌(Escherichia coli)之生理現象。以往的研究顯示,重組蛋白質大量表達時,會引起寄主細胞(host cell)的生長抑止(growth inhibition)。本研究發現在生長抑止的同時會伴隨著細胞溝通的現象。而且細胞會將質體排斥到細胞外或降解質體,造成質體大量流失。
本研究共分成兩部分,一是用重組質體(pJL),來探討細胞溝通的現象。另一是rpoD互補系統(VJS632rpoD285/ pTRL) 為實驗對象。兩重組質體皆可受IPTG所誘導,使lacZ基因表現,生產半乳糖苷酶(β-galactosidase)。
研究中我們試著以標記性細胞與帶質體細胞(VJS632/pJL)的混合培養、兩區發酵槽的共同培養,及以conditioned medium培養等三種方法去證明細胞溝通。實驗結果發現,當過度表達生產重組蛋白質時,帶質體的細胞確實會抑制不帶質體細胞的生長,我們推論這是因為細胞溝通引起的。
另一部分,藉以rpoD互補系統去探討質體流失的機制。此系統宿主大腸桿菌(VJS632rpoD285)本身為rpoD基因缺陷,而對溫度產生敏感,故無法於42℃的環境下生長;而以一質體(pTRL)帶有正常的rpoD基因作為互補。因此帶有質體的細菌,方能在42℃的環境下生長。實驗結果發現,以rpoD互補系統在42℃下培養,系統在無誘導下,確實可維持極高的質體穩定度;但當系統在過度表達時,帶質體細胞的比例卻大幅下降。因此我們推測在過度表達生產重組蛋白時,質體會大量的被排斥到細胞外,造成質體大量流失,而非單純僅因分裂損失所造成。
論文英文摘要:In this thesis, we study physiology of Escherichia coli during protein overexpression as plasmid is the vector. Evidences for cell communication and plasmid pushing-out during protein overexpression are found in this study.
Mixed culture, co-culture, and condition medium were used to demonstrate cell communication. We found the growth rate of plasmid-free cell will be inhibited by plasmid-carrying cell during protein overexpression, which may be the proof of cell communication.
VJS632rpoD285/pTRL is rpoD complementary system. The host cell VJS632rpoD285 itself has a defect in rpoD gene, making it sensitive to high temperature, thus it can not survive at 42℃. The complementary plasmid, pTRL carries a functional rpoD gene. Hence the mutant cell containing the plasmid can survive at 42℃. Without induction, the experimental results show that it keeps a high level of plasmid stability at 42℃. However, when recombinant proteins were overexpressed, the ratio of plasmid-carrying cell greatly decreased. Hence we infer that protein overexpression triggers host cells to push out or destroy plasmid. Plasmid instability is not caused by segregation loss only and physiological mechanism may be involved.
論文目次:目 錄

中文摘要……………………………………………………………………………..… i
英文摘要……………………………………………………………………………..… ii
致謝……………………………………………………………………………………..iii
目錄………………………………………………...…………………………………...iv
表目錄……………………………………………...………………………………...…vi
圖目錄……………………………………………...……………………………..........vii
第一章 緒 論 1
1.1 前言 1
1.2 研究動機 4
第二章 文獻回顧 5
2-1 細胞溝通 5
2-2 質體穩定性 8
第三章 材料與方法 10
3.1.1菌種與質體 10
3.1.2藥品與材料 12
3.2 實驗儀器與設備 14
3.3 研究策略 16
3.3.1 菌種保存 17
3.3.2 培養基組成 18
3.4構築標記性細胞 19
3.4.1 P1溶胞產物(lysate)的製備 20
3.4.2 轉導作用(transduction) 20
3.5 塗佈方式下的生長速率分析 21
3.6兩區發酵槽培養 22
3.6.1 前處理 22
3.6.2 生長速率量測 23
3.7質體純化 24
3.7.1鹼性純化法 24
3.7.2 質體純化組(kit)純化 24
3.8轉形作用 25
3.8.1化學法 25
3.8.2電轉殖法 26
3.9 批次培養 27
3.10 過度表達生產重組蛋白質之批次培養 27
3.11 質體穩定性分析 27
3.12 複製培養 (replica plating) 29
第四章 帶質體細胞與不帶質體細胞間的溝通 30
4.1 初步實驗結果 (preliminary data) 30
4.1.1 生長抑制 30
4.1.2 質體不穩定 31
4.1.3混合生長比較 32
4.2 混合培養 ( mixed culture ) 35
4.2.1 標記性細胞的構築 35
4.2.2 以塗佈方式求在無誘導下之生長速率 38
4.2.3 以塗佈方式求在過度表達下的生長速率 40
4.3 共同培養 ( co-culture ) 42
4.3.1 培養條件的設定 42
4.3.2 過度表達下的生長速率比較 43
4.3.3 以IPTG假設為訊號因子下的生長分析 45
4.4 於Conditioned medium下的培養 47
第五章 質體流失機制的探討 49
5.1 rpoD系統無誘導於42℃下的質體穩定性分析 49
5.2 rpoD系統過度表達於42℃下的質體穩定性分析 52
5.3複製培養 54
第六章 結論 55
參考文獻 57
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53. 陳建超,(2002)〝生產重組蛋白質對質體不穩定之影響〞, 國立台北科技大學化學工程研究所碩士論文。
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