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研究生:游大慶
研究生(外文):Da-Chin You
論文名稱:生長條件及誘導溫度對大腸桿菌生產白細胞介素-20之影響
論文名稱(外文):The effect of cell condition and temperture for induction on the prodution of interleukin-20 by Escherichia coli
指導教授:陳特良
指導教授(外文):Teh-Liang Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
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:2002
畢業學年度:90
語文別:中文
論文頁數:46
中文關鍵詞:大腸桿菌白細胞介素誘導溫度
外文關鍵詞:escherichia coliinterleukintemperature
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  本研究以基因重組Eshrechia coli.BL21(DE3)/(pET43.1a)生產白細胞介素-20( interleukin-20 ),此蛋白質可用於乾癬(psoriasis)之治療研究。研究發現此菌株最適生長溫度為42oC,不適合在鹼性條件下生長,溶氧值維持在20%以上即已足夠菌體之生長。在同一生長溫度下誘導之最佳溫度為37oC。在生長溫度為42oC下操作,並改變其誘導後之溫度,所得最高目標蛋白質濃度為相當於BSA 0.42 g/L。降低生長溫度為37OC,菌體濃度雖僅能達14.3 g/L,但目標蛋白質產量卻可達0.89 g/L ﹔實驗後發現生長溫度37OC之蛋白質分解酵素產生較42oC少,而等量之蛋白質分解酵素在本實驗所改變的誘導溫度範圍內活性差不多,故推測蛋白質分解酵素是降低生長溫度後蛋白質產量提高之主因。本菌株亦可添加乳糖誘導,實驗發現,添加10 g/L乳糖誘導,目標蛋白質產量極微小,若添加20 g/L乳糖誘導則目標蛋白質產量可達1.135 g/L,比產率為0.05 g/g cell比利用IPTG誘導之比產率(0.062 g/g cell)略低。經電泳分析發現本菌株在此研究系統中有包涵體的產生,勢必也會對蛋白質產量造成影響。
  The present research used a recombinant strain, Escherichia coli BL21(DE3)/(PET43.1a),to produce interleukin-20 which can be used to treat and study with pso-riasis. With this research we found that the most suitable temperature for the growth of this strain is 42oC, and it can’t grow well in basic condition ,and the DO 20% is enough for the growth of the srtain.37oC is the best temperature for production at the same growth temperature. With the 42oC growth temperature, the highest protein concentration it can reach is 0.42 g/L. When we lower the growth temperature to 37oC,in spite of cell density 14.3 g/L, the target protein concentration can rise to 0.898 g/L. At this study, the protease concentration is higher at 42oC growth temperature than 37oC, and the protease activity is about the same within all the production temperature range. According to the above result, the main reason for the rise of the target protein concentration is the protease concentration. This strain can also be induced with lactose. After batch fermentation, we found that 10 g/L lactose is not enough for this strain to express significant amount of target protein. When we used 20 g/L lactose for induction, 1.135 g/L of our target protein can be produced. And its specific productivity is 0.05 g/g cell, which is a little lower than the value 0.062g/g cell achieved when induced with 1 mM IPTG. It will affect the production of our target protein that this strain can produce inclusion body .
中文摘要 ……………………………………………………………Ⅰ
英文摘要 …………………………………………………………..Ⅱ
誌謝………………………………………………………………….Ⅲ
總目錄……………………………………………………………….Ⅳ
內文目錄…………………………………………………………….Ⅴ
圖目錄……………………………………………………………….Ⅶ
表目錄……………………………………………………………….Ⅸ
內文目錄
第一章緒論
1-1前言…………………………………………………………… 1
1-2白細胞介素-20之簡介……………………………………….3
1-2-1白細胞介素-10家族的新成員………………………….3
1-2-2白細胞介素-20的主作用目標是皮膚………………….3
1-2-3白細胞介素-20接受體與乾癬之關聯………………….4
1-3銀屑病(乾癬)之簡介………………………………………….5
1-4研究動機……………………………………………………….6
第二章材料與方法
2-1菌株及藥品
2-2-1菌株……………………………………………………….7
2-1-2藥品……………………………………………………….8
2-2儀器與裝置…………………………………………………….9
2-3實驗方法
2-3-1菌種之培養與保存………………………………………10
2-3-2醱酵實驗…………………………………………………10
2-3-3 溶氧電極之校正……………………………………….11
2-4分析方法
2-4-1菌體濃度…………………………………………………11
2-4-2 白細胞介素-20之分析…………………………………11
2-4-3 葡萄糖濃度……………………………………………..16
2-4-4 質體穩定性之分析……………………………………..18
2-4-5 醋酸濃度之分析………………………………………..18
2-4-6 蛋白質分解酵素活性之分析………………………….20
2-4-7包涵體之分析方法…………………………………….22
第三章結果與討論
3-1 培養基組成………………………………………………….23
3-2 轉速與溶氧值對菌體生長之影響………………………….23
3-3溫度對菌體生長之影響……………………………………..23
3-4 pH值對菌體生長之影響…………………………………….26
3-5以42oC之溫度生長並改變誘導後之溫度………………….26
3-6 降低生長溫度並以IPTG及乳糖誘導生產白細胞
介素-20………………………………………………………30
3-7 不同生長溫度下誘導後目標蛋白質產量之比較…………..39
第四章結論………………………………………………………..42
參考文獻…………………………………………………………….44
圖目錄
圖2-1 本菌株之基因限制圖譜……………………………………….7
圖2-2 批次發酵槽裝置圖……………………………………………12
圖2-3菌體濃度在600 nm下之檢量線……………………………..14
圖2-4 BSA濃度在595 nm下之檢量線………………………………17
圖2-5 葡萄糖濃度在550 nm下之檢量線…………………………..19
圖2-6 醋酸濃度之檢量線…………………………………………….21
圖3-1不同轉速下菌體之生長曲線………………………………….24
圖3-2不同溶氧值下菌體之生長曲線……………………………….25
圖3-3不同溫度下菌體之生長曲線………………………………….27
圖3-4不同pH值下菌體之生長曲線………………………………..28
圖3-5誘導後改變溫度菌體之生長及蛋白質生產情形…………….29
圖3-6誘導溫度37o在不同下反應溫度下測得之蛋白質分解酵素之活性……………………………………………………………….31
圖3-7不同誘導溫度在37oC下反應測得之蛋白質分解酵素
之活性………………………………………………………….32
圖3-8不同誘導溫度在42oC下反應測得之蛋白質分解酵素之
活性…………………………………………………………….33
圖3-9不同誘導溫度在32oC下反應測得之蛋白質分解酵素之
活性……………………………………………………………34
圖3-10在37oC下以乳糖及IPTG誘導菌體之生長曲線及蛋白質之生產………………………………………………………….35
圖3-11在37oC下以乳糖及IPTG誘導並改變溫度至32oC之菌體生長曲線及蛋白質之生產……………………………………………….37
圖3-12在37oC下以乳糖誘導菌體之生長曲線及蛋白質之
生產……………………………………………………….38
圖3-13不同生長溫度在37oC下反應測得之蛋白質分解酵素之
活性………………………………………………………….41
表目錄
表2-1 Modified LB 之成分組成………………………………….13
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