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研究生:郭匡甫
研究生(外文):Kuang-Fu Kuo
論文名稱:誘導後比生長速率對基因重組大腸桿菌生產白細胞介素20號的影響
論文名稱(外文):Effect of the Post-Induction Specific Growth Rate on the Production of Interleukin-20 by recombinant E. 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:51
中文關鍵詞:誘導後比生長速率基因重組大腸桿菌白介素20號比產量批式饋料
外文關鍵詞:Post-induction specific growth rateIL-20 Yieldfed-batchrecombinant E. coli
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  目前利用基因重組大腸桿菌生產外來蛋白質之醱酵方法分成兩階段,首先以批次培養提高菌體濃度,其次以饋料批次饋入新鮮培養基與誘導劑,以誘導外來基因表現。由於以往的文獻發現在批次培養中大腸桿菌比生長速率是影響異種蛋白質產量的重要參數,因此本研究之目的在於探討誘導後細胞比生長速率對於重組蛋白白細胞介素20號(IL-20)生產之影響。研究發現,本研究所使用之宿主載體系統Escherichia coli BL21 (DE3)/pET-43a產生的醋酸僅在2 g/L以下,所以醱酵系統並無醋酸累積之問題。其次內涵體的累積亦不明顯,IL-20主要是以可溶性狀態存在於細胞內。其次,以平均比生長速率0.103 h-1進行饋料可得到最高的比產量(109 mg/g cell),此結果顯示誘導後比生長速率對比IL-20產量之影響存在一最適值。將實驗數據進一步分析,若以蛋白質比生產速率之觀點,將平均比生長速率控制在0.23 h-1可得到IL-20最高比生產速率16.4 mg/(g cell)•h。
The method for producing recombinant proteins by Escherichia coli consists of two stages. The first was to increase cell density at the batch-cultured stage; and the second was to feed fresh medium with inducer using fed-batch operation for inducing the expression of foreign gene. The literatures had documented that the cell growth rate is a major factor affecting the yield of recombinant proteins. Therefore, the objective of this study was to investigate the effect of cell growth rate of post-induction on the production of the recombinant protein interleukin-20 (IL-20). The results indicated that the host-vector system E. coli BL21(DE3) produced little acetic acid (below 2 g/L), thus the fermentation system was not influenced by the accumulation of acetate. The formation of inclusion bodies were not obvious, most of the IL-20 existed as soluble form inside the cells. In addition, the highest specific IL-20 yield (109 mg/g cell) was obtained when the average growth rate was 0.103 h-1. From the experimental data, the highest specific production rate of IL-20 was estimated as 16.4 mg/(g cell)•h if the average cell growth rate could be controlled at 0.23 h-1.
總目錄
頁數
中文摘要……………………………………………………………………………………I
英文摘要…………………………………………………………………..………………Ⅱ
總目錄……………………………………………………………………………………..Ⅲ
表目錄 ……………………………………………………………………………………Ⅳ
圖目錄 ……………………………………………………………………………………Ⅴ
符號……………………………………………………………………………………....Ⅵ
第一章 緒論 …………………………………………………………………….………...1
1-1前言 ……………………………………………………………………….……...1
1-2質體穩定性………………………………………………………………………..1
1-3重組蛋白的控制和誘導……………………………………….………………….2
1-4影響高菌體濃度醱酵的原因……………………………………………………..4
1-4-1醱酵策略…………………………………………………………………4
1-4-2葡萄糖的添加……………………………………………………………5
1-4-3醋酸的抑制………………………………………………………………5
1-5研究動機………………………………………………………………….……….6
1-5-1比生長速率對蛋白質產量的影響……………………………………….7
1-5-2IPTG對菌體的影響……………………………………………………...7
1-5-3內涵體的形成…………………………………………………………….8
1-6研究目標………………………………………………………….……………….9
1-7文獻參考………………………………………………………………….…….....9
第二章 實驗材料和方法………………………………………………………………11
2-1菌株和藥品………….…………………………………………………………….11
2-1-1菌株………………………………………………………………………11
2-1-2藥品………………………………………………………………………11
2-2 儀器與裝置………….…………………………………………………….……14
2-3 實驗方法………………………………………………………………………….14
2-3-1 菌體的活化前培養和保存…………….………………………………14
2-3-2 醱酵實驗………………………………………………………………17
2-4分析方法…………………………………………………………………………..21
2-4-1菌體濃度分析方法……………………………………………………....21
2-4-2葡萄糖濃度的測定……………………………………………………....21
2-4-3醋酸檢量線...……………………………………………………………23
2-4-4質體穩定性………………………………………………………………23
2-4-5白細胞介素20號的定性分析…………………………………………...27
2-4-6 蛋白質的定量分析……………………………………………………...31
2-5 饋料批式改變比生長速率…………….………………………………………..31
2-5-1 饋料速率的預估………………………………………………………...31
2-5-2 比生長速率的計算……………………………………………………...33
第三章 結果與討論……………………………………………………………………..35
3-1 固定速率饋料所得不同比生長速率的情形………...…………………………35
3-2 誘導後的饋料批式醱酵數據……………………………..…………………….35
3-3 質體穩定性和誘導後比生長速率的關係….………..…………………………38
3-4 白細胞介素20號的比產量和比生長速率的關係…...……..…………………44
3-5 白細胞介素20號的比生產速率和比生長速率的關係……………………….44
3-6 結論……………………………………………………………………………...47
參考文獻……………………………………………..……………………………………48
表目錄
頁數
表 2-1 儀器與設備…..……………….………………………………………………….15
表 2-2 固態LB培養基的成分……….…..……………………………………………..18
表 2-3主培養所使用的合成培養基…………………………………………………….19
表 2-4氣相層析儀的操作條件.........................................................................…………25
表 2-5電泳溶液成份……………………………….……………………………………29
表 3-1各誘導後比生長速率對質體穩定性的影響…………….………………………45
圖目錄
頁數
圖1-1誘導型的啟動子的誘導機制圖…………………………….………………….…...3
圖2-1質體pET-43a的限制圖譜.…………………………………………………..……..12
圖2-2醱酵裝置和控制裝置圖...…………………………………………………..……..20
圖2-3菌體濃度檢量線…………………………………………………..………….……22
圖2-4葡萄糖濃度檢量線……………………………………………..…………….……24
圖2-5醋酸的檢量線……………………………………………………..……………….26
圖2-6目標蛋白質白細胞介素20號的電泳圖………………………………………….30
圖2-7牛血清蛋白濃度的檢量線…..………………………………….……….………....32
圖3-1誘導後比生長速率μ= 0.407 h-1…………………………………………………..36
圖3-2誘導後比生長速率μ= 0.331 h-1…………………………………………………..37
圖3-3誘導後比生長速率μ= 0.23 h-1………………………………………………..…..39
圖3-4誘導後比生長速率μ= 0.183 h-1…………………………………………………..40
圖3-5誘導後比生長速率μ= 0.103 h-1…………………………………………………..41
圖3-6誘導後比生長速率μ= 0.056 h-1…………………………………………………..42
圖3-7各誘導後比生長速率下的目標蛋白質比產量……………………….…………..45
圖3-8各誘導後生比長速率下的目標蛋白質比產率…………………………………...46
符號
F (mLh-1)………………………………………….…………………..………....體積流速
S (gL-1)……………………..………………………………………………………基質濃度
S0 (gL-1)………………………………………………………………醱酵開始的基質濃度
t (h)…………………………….……………………………………………….…….....時間
Vf (mL)…………………………………………………………....….….醱酵結束時的體積
V0 (mL)…….……………………………………………………...….…醱酵開始時的體積
X (gL-1)…………………………………………………………………..…….…..菌體濃度
X0 (gL-1)……………………………………………………………醱酵開始時的菌體濃度
YX/S (g DCW/g Glucose)………………….………………………………..…..…..產量係數
μ (h-1)………………………………………….……………………………….比生長速率
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