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研究生:邱倉德
研究生(外文):chiu tsang-te
論文名稱:基因重组技術促進水母冷光酶之活性與產量
論文名稱(外文):Fusion Renilla reniformis luciferase with VHb to enhance its activity and productivity
指導教授:李振綱李振綱引用關係
指導教授(外文):cheng-kang lee
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:126
中文關鍵詞:水母冷光酶質體穩定度透明顫菌血紅蛋白
外文關鍵詞:Renilla reniformis luciferaseVHb
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水母冷光酶(Renilla reniformis luciferase;RLuc)為可以催化腔腸素(coelenterazine)氧化而產生光的酵素,此發光機構簡單極具商業價值,本論文是研究如何提高水母冷光酶產量和活性為目的。
首先我們將RLuc基因建構在多複製數質體上成為pTXB1-RLuc,並做為表現之控制組,再將穩定高複製數低的mini-F質體做為表現載體,利用基因重組技術使其上帶有許多份數的RLuc 基因片段,成功地建構出一到三個RLuc 基因份數的質體:pBAC-1×RLuc、pBAC-2×RLuc、pBAC-3×RLuc,經過表現及質體穩定度測試後,發現帶有pBAC-3×RLuc的蛋白質表現量為882.70μg/ml較其它兩者的產量多,但仍少於pTXB1-RLuc的1382.62μg/ml,然而其穩定性在誘導後十二個小時質體懷有率仍高達百分之九十七,遠大於pTXB1-RLuc的百分之四。
水母冷光酶的活性受環境影響很大,在100mM磷酸緩衝液中活性最高,pH7為最適的酸鹼度,最佳反應溫度在37℃。另一方面,金屬螯合層析(IMAC)所使用的脫附劑Imidazole會抑制RLuc的活性,以UF膜過濾去除Imidazole,其活性可恢復為原來的83.21%。
由於水母冷光酶反應需要氧氣的參與,而透明顫菌血紅蛋白(Vitreoscilla hemoglobin; VHb)具有吸附氧氣的能力,因此本論文也利用蛋白質融合(Protein fusion)技術,將VHb融合在RLuc上,發現菌體表現融合VHb的RLuc其生長速率和最終濃度都高於對照組;在微氧反應條件下,在N端及C端融合有VHb的RLuc活性也高於RLuc的1.98倍和2.06倍。
Renilla riniformis luciferase (RLuc) catalyzes coelenterazine oxidation to produce light. RLuc gene was expressed in E. coli to facilitate the large-scale production of RLuc. RLuc gene of 1 to 3 copies were cloned to the very stable F-plasmid and named as pBAC-1xRLuc, pBAC-2xRLuc, and pBAC-3xRLuc. The expression of RLuc was driven by the strong T7 promoter. The expression level of pBAC-3xRLuc was higher than two others, but lower than the pET expression vector. RLuc activity was strongly inhibited by imidazole employed in IMAC. Removing imidazole resumed RLuc activity to about 83.21 percentage. O2 plays a very important role in the RLuc catalyzed oxidation. Vitreoscilla hemoglobin (VHb) is able to bind O2. RLuc fused with VHb enhanced its activity by 2 fold. Besides, the fused VHb still retained its activity which also enhanced the cells growth. As a consequence, the productivity of RLuc activity was enhanced bye fusing with VHb.
目 錄
第一章 緒論
1.1 研究背景 P1
1.2 研究內容與目的 P2
第二章 文獻回顧
2.1 生物冷光 P4
2.2 水母冷光酶 P6
2.3 F質體 P8
2.4 提高蛋白質產量的策略 P9
2.5 質體穩定度 P11
2.6 細菌的血紅素 P13
2.7 利用VHb改善細胞生長及產率 P15
2.8 融合蛋白的技術 P16
第三章 實驗
3.1 實驗流程 P18
3.2 表現載體建構的方法 P18
3.3 實驗材料 P35
3.4 實驗藥品 P38
3.5 實驗設備 P40
3.6 實驗步驟 P42
第四章 結果與討論
4.1 含多份數RLuc基因之mini-F質體其冷光酶表現量及其質體穩定度 P79
4.2 Luciferase的活性分析研究 P92
4.3 融合VHb對RLuc其酵素活性及促進菌株生長的影響 P96

第五章 結論與建議
5.1 結論 P107
5.2 研究建議 P108
參考文獻 P110
附 錄 P116
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