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研究生:洪金榮
研究生(外文):Chin-Jung Hung
論文名稱:以基因重組大腸桿菌生產白細胞介素20號:誘導劑添加方式之研究
論文名稱(外文):Methods for Inducer Addition in the Production 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:2003
畢業學年度:91
語文別:中文
論文頁數:60
中文關鍵詞:基因重組大腸桿菌重組蛋白質誘導白細胞介素20號
外文關鍵詞:Escherichia coliInterleukin 20IPTGInduction
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目前以基因重組大腸桿菌生產異種蛋白質常見之醱酵策略,是以批次培養提高菌體濃度,再以饋料批次方式添加新鮮培養基與誘導劑異丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside, IPTG);所以,過去的文獻大多以饋料的方式對於重組蛋白質生產之影響做為研究主題。然而到目前為止,並沒有研究人員深入討論直接影響蛋白質表現的IPTG之添加依據。有鑑於此本研究以Escherichia coli BL21 (DE3)/pET-43a為材料,探討白細胞介素20號(IL-20)的生產時,應根據何種原則添加IPTG。預先設定的IPTG之添加原則包括:(1)以固定濃度方式為之、(2)以誘導時的菌體量為依據、(3)以最終的菌體量為依據、(4)以誘導後菌體的增加量為依據。
研究結果顯示IL-20最終比產量與IPTG的添加量並無對應關係,但事實上重組蛋白質的最終比產量應該與IPTG添加量和菌體量都有關係,因此預先設定的四種原則均不適合做為IPTG之添加依據。然而,實驗數據卻顯示IL-20之比生產速率隨著IPTG添加濃度的增加而提高、誘導後第一小時內質體安定性與IPTG添加濃度成反比。除了IPTG的量以外,還有其他因素會造成重組蛋白質產量的差異,但如能以適當的IPTG濃度添加並配合養份的持續進料,可能會維持IL-20的生產。
The common strategy for producing recombinant proteins by Escherichia coli consists increasing cell density at the batch-cultured stage and feeding fresh medium with inducer at the fed-batch stage. For the past years, the effect of feeding strategy for the production of recombinant proteins had been studied extensively. However, the rule for adding inducer (isopropyl-□-D-thiogalactopranoside, IPTG), influences the production of foreign proteins, had never been discussed yet. Accordingly, the objective of this study was to investigate the rule of IPTG addition employing E. coli BL21 (DE3)/pET-43a as the IL-20 producing strain. The proposed rules for the addition of IPTG were according to: (1) constant IPTG concentration, (2) the cell mass at induction, (3) the cell mass at the end of fermentation, and (4) the increase of cell mass after induction.
The results first showed that there was no correlation between the amount of IPTG addition and the specific yield of IL-20. In fact, the specific yield of recombinant protein was affected by the amount of IPTG and cell mass. Therefore, the addition of IPTG can not determined by the rules proposed before. However, the data indicated that the specific production rate of IL-20 increased with the increase of IPTG concentration. Besides, the plasmid stability decreased in the first hour after induction as the increase of IPTG concentration. In addition to IPTG, the yield of recombinant protein was affected by other factors. However, it was proposed that the production of IL-20 might continue if both appropriate concentration of IPTG and fresh medium were fed to the fermentation system.
總目錄
頁數
中文摘
要……………………………………………………………………………..I
英文摘要…………………………………………………………………………………..II
誌謝……………………………………………………………………………………….III
總目錄……………………………………………………………………………………..Ⅳ
表目錄……………………………………………………………………………………..VI
圖目錄…………………………………………………………………………………….VII
第一章 緒論
1-1 前言…………………………………………………………………………..…1
1-2 誘導機制……………………………………………………………………..…2
1- 3白細胞介素20號……………………………………………………………….....4
1-4 研究動機……………………………………………………………………….....6
第二章 實驗材料與方法
2-1 菌株及藥品
2-1-1 菌株……………………………………………………………………..8
2-1-2 藥品……………………………………………………………………..8
2-2 儀器與裝置…………………………………………………………………….10
2-3 實驗方法
2-3-1 菌種保存………………………………………………………………..12
2-3-2 培養基…………………………………………………………………..12
2-3-3 醱酵實驗………………………………………………………………..14
2-4 分析方法
2-4-1 菌體濃度分析方法……………………………………………………..16
2-4-2 葡萄糖濃度之分析……………………………………………………..16
2-4-3 醋酸濃度的測定………………………………………………………..19
2-4-4 質體穩定性之分析……………………………………………………..22
2-4-5 十二烷基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE)………………..22
2-4-6白細胞介素20號濃度之分析…………………………………………...25
2-5 實驗參數之定義………………………………………………………………..26
第三章 結果與討論
3-1 以不同濃度IPTG誘導對於IL-20生產之影響︰1倍濃度R medium……......29
3-2 IPTG添加量對於IL-20生產之影響︰0.5倍濃度R medium………………....34
3-3 IPTG添加量對於IL-20生產之影響︰1.5倍濃度R medium………………....36
3-4 IPTG添加對於IL-20比生產速率與最終比產量之影響……………………...47
3-5 IPTG添加量對於質體穩定性的影響…………………………………………..47
第四章 結論……………………………………………………………………………..53
第五章 未來展望………………………………………………………………………..55
參考文獻………………………………………………………………………………...56
自述……………………………………………………………………………………...60
表目錄
頁數
表2-1 醱酵培養基R medium之組成........................................13
表3-1 不同IPTG添加量對於誘導後細胞比生長速率、IL-20比生產速率、IL-20
比產量與質體安定性之影響(1倍R medium)…………………………….…..33
表3-2 不同IPTG添加量對於誘導後細胞比生長速率、IL-20比生產速率、IL-20
比產量與質體安定性之影響(0.5倍R medium)……………………………...40
表3-3 不同IPTG添加量對於誘導後細胞比生長速率、IL-20比生產速率、IL-20
比產量與質體安定性之影響(1.5倍R medium)……………………………...46
圖目錄
頁數
圖1-1 乳糖操縱基因組(lac operon)之誘導調節示意圖………………………………3
圖1-2 乳糖分解成半乳糖及葡萄糖之過程……………………………………………..5
圖2-1 醱酵實驗裝置…………………………………………………………………….15
圖2-2 實驗流程………………………………………………………………………….17
圖2-3 菌體檢量線……………………………………………………………………….18
圖2-4 葡萄糖檢量線…………………………………………………………………….20
圖2-5 醋酸濃度檢量線………………………………………………………………….21
圖2-6 牛血清蛋白(BSA)檢量線………………………………………………………..24
圖2-7 IL-20電泳圖……………………………………………………………………..27
圖2-8 IL-20比生產速率之定義………………………………………………………..28
圖3-1 兩階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第8小時加入
400 ml R medium(含最終濃度1.0 mM IPTG)………………………………….30
圖3-2 兩階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第8小時加入
400 ml R medium(含最終濃度1.5 mM IPTG)………………………………….31
圖3-3 兩階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第8小時加入
400 ml R medium(含最終濃度2.0 mM IPTG)………………………………….32
圖3-4 R培養基濃度減半之菌體生長曲線……………………………………………..35
圖3-5 兩階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第6小時加入
400 ml R medium(含最終濃度1.5 mM IPTG)………………………………….37
圖3-6 兩階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第6小時加入
400 ml R medium(含356 mg IPTG,視誘導初始菌量添加)……………...…38
圖3-7 兩階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第6小時加入
400 ml R medium(含290 mg IPTG,視誘導後細胞增加量添加)…………...39
圖3-8 R培養基濃度增加一半之菌體生長曲線……………………………………....41
圖3-9 三階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第9小時加入
400 ml R medium(含最終濃度1.5 mM IPTG)……………………………..….43
圖3-10 三階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第9小時加
入400 ml R medium(含893 mg IPTG,視誘導初始菌量添加)……………..44
圖3-11 三階段批次醱酵中各實驗參數與醱酵時間之關係:醱酵時間第9小時加
入400 ml R medium(含804 mg IPTG,視誘導後細胞增加量添加)………..45
圖3-12不同的IPTG添加依據對IL-20比生產速率之影響…………….……..….....48
圖3-13不同的IPTG添加依據對IL-20最終比產量之影響…………….………….....49
圖3-14 IPTG添加量對於質體穩定性之影響……………………………………......50
圖3-15不同的IPTG添加依據對誘導後第一小時內質體穩定性之影響.…………....52
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