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系統識別號 U0002-0608201022091400
中文論文名稱 人類顆粒細胞增生因子基因密碼在酵母菌中使用頻率的研究
英文論文名稱 Codon Optimization and Expression of Human Granulocyte Colony Stimulating Factor
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 98
學期 2
出版年 99
研究生中文姓名 汪恩慶
研究生英文姓名 En-Ching Wang
學號 697160595
學位類別 碩士
語文別 中文
口試日期 2010-07-12
論文頁數 72頁
口試委員 指導教授-簡素芳
委員-莊子超
委員-簡敦誠
委員-簡素芳
中文關鍵字 人類顆粒細胞增生因子  密碼偏好 
英文關鍵字 Human Granulocyte Colony Stimulating Factor  hG-CSF  Codon usage bias 
學科別分類 學科別自然科學化學
中文摘要 人類顆粒細胞增生因子(Human Granulocyte Colony Stimulating Factor;hG-CSF)能刺激嗜中性白血球前驅細胞生長,並促進其增殖、分化,同時具有促進嗜中性白血球由骨髓釋出及增強成熟嗜中性白血球的機能,它被廣泛的應用在癌症化療和骨髓移植後,所引起的嗜中性白血球缺乏症(neutropenia)。
酵母菌Pichia pastoris是常用於表現重組蛋白的宿主細胞之一,具有將蛋白質正確地折疊、轉譯後修飾以及將蛋白分泌至胞外等優點。在我們實驗室利用發酵槽誘導酵母菌可以獲得hG-CSF約71 mg/L。
對宿主細胞而言的密碼使用頻率較高的基因可能會使蛋白產量有所提昇,因此本實驗依據酵母菌的密碼偏好,設計並合成出最佳化密碼的hG-CSF基因(syn hG-CSF),利用分析CAI值預測此段基因在大腸桿菌E. coli和酵母菌P. pastoris之中會有較高的蛋白表現,之後分別轉殖到大腸桿菌BL21(DE3)跟酵母菌SMD1168之中。
目前已成功獲得大腸桿菌轉型株,在37℃下,使用搖瓶的方式加入IPTG誘導4個小時表現syn hG-CSF,以SDS-PAGE及西方點墨法(Western blotting)分析,發現所表現的蛋白分子量符合在22 kDa的位置,在500毫升的菌液當中大約有100毫克目標蛋白。
英文摘要 The recombinant human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that has been widely used for the treatment of neutropenia after chemotherapy and bone marrow transplantation. It can stimulate both proliferation and differentiation of neutrophils.
Pichia pastoris has been extensively used as a cellular host for recombinant protein expression. It’s advantages are posttranslational modification, protein folding correction and protein secretion to the medium. In our laboratory, the expression of hG-CSF in P.pastoris,about 71 mg/L of recombinant protein in bioreactor.
In theory, the higer the codon usage bias by th host cell, the more improved yield of protein.Therefore in this study, we designed and synthesized a new hG-CSF gene by using yeast preferred codon.The use of CAI values predicted this gene would yield higher expression level when transferred into E. coli BL21(DE3) and P.pastoris SMD1168.
Up to the present, we have successfully cultured the colony of E. coli. after 4 hours of induction by IPTG at 37℃, the expression of the recombinant protein was confirmed by SDS-PAGE and Western blotting. The amount of recombinant protein was 100 mg in 500 ml LB medium approximately, and molecular weight was as predicted at 22 kDa corresponding to the commercial standard.
論文目次 封面內頁
口試委員審議通過簽名表
授權書
致謝
中文摘要 I
英文摘要 II
目錄 III
圖表目錄 V

第一章 緒論
第一節 前言 1
第二節 人類顆粒細胞增生因子 2
第三節 生物細胞內蛋白質合成之密碼使用偏好 5
第四節 原核細胞表現系統 7

第二章 實驗材料、設備
第一節 實驗材料 14
第二節 實驗設備 17
第三節 培養基配置 18

第三章 實驗方法
第一節 hG-CSF synthesize gene基因選殖 20
第二節 pET25b表現質體和syn hG-CSF基因之重組 33
第三節 基因表現 37
第四節 重組蛋白質之純化 43

第四章 實驗結果
第一節 基因建構 45
第二節 確認和純化大腸桿菌所表現之重組蛋白 47

第五章 結論與未來展望
第一節 實驗結論 63
第二節 未來展望 64

第六章 參考資料 69


表 1.1 Triplet codon與胺基酸的對應表 13

表3.1 PCR(gene extension)反應之各項成分 24
表3.2 PCR(gene amplification)反應之各項成分 24
表3.3 PCR(gene extension)反應條件 25
表3.4 PCR(gene amplification)反應條件 25
表3.5 TA ligation之各項成分 29
表3.6 pGEM::hG-CSF DNA以限制酶(NcoI、NotI)水解反應組成 32
表3.7 pET25b 質體DNA以限制酶(NcoI、NotI)水解之反應組成。 33
表3.8 pGEM::syn hG-CSF以限制酶(NcoI、NotI)水解之反應組成。 34
表3.9 接合反應溶液。 35
表3.10 pET25b::syn hG-CSF以限制酶(NcoI、NotI)水解之反應組成。 36

表5.1 生物技術工業常用的蛋白質表現系統 67

圖 1.1 不同增生因子對造血幹細胞分化圖 9
圖 1.2 G-CSF蛋白質分子之三級結構 10
圖 1.3 hG-CSF與其他相似結構之細胞激素比較圖 11
圖 1.4 原核細胞表現系統蛋白質調控機制 12

圖 2.1 pET25b(+)表現載體 19

圖 3.1 以酵母菌的codon usage所擬出之hG-CSF DNA序列及其對應胺基酸 20
圖 3.2 依照所擬的hG-CSF DNA序列來設計之primer位置圖 22
圖 3.3 PCR反應說明圖 23
圖 3.4 TA vector:pGEM®-T Vector質體圖 28
圖 3.5 轉漬槽示意圖 42

圖 4.1 hG-CSF(上) 與syn hG-CSF(下)DNA序列比對圖 50
圖 4.2 hG-CSF(上)與syn hG-CSF (下)DNA轉譯出的胺基酸序列圖 51
圖 4.3 a) hG-CSF
(b) syn hG-CSF在大腸桿菌中的CAI值 52
圖 4.4 洋菜膠電泳分析PCR(gene extension)之結果 53
圖 4.5 洋菜膠電泳分析PCR(gene amplification)之結果 54
圖 4.6 TA cloning 藍白篩選 55
圖 4.7 洋菜膠電泳分析pGEM::syn hG-CSF DNA 56
圖 4.8 (a)洋菜膠電泳確認pET25b::syn hG-CSF DNA
(b)pET25b::syn hG-CSF質體圖 57
圖 4.9 誘導重組蛋白表現 58
圖 4.10 誘導時間和誘導蛋白表現量的關係圖 59
圖 4.11 破菌前後之SDS-PAGE 和 Western blotting 60
圖 4.12 利用市售hG-CSF進行蛋白質定量 61
圖 4.13 純化蛋白(inclusion body)之SDS-PAGE 和 Western blotting 62

圖5.1 異源蛋白質分泌至胞外示意圖 68
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