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系統識別號 U0002-3107200523461100
中文論文名稱 酵母菌(Pichia pastoris)的基因表現
英文論文名稱 Gene Expression in Yeast(Pichia pastoris)
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 93
學期 2
出版年 94
研究生中文姓名 秦嘉志
研究生英文姓名 Chia-Chi Chin
學號 692171076
學位類別 碩士
語文別 中文
口試日期 2005-06-15
論文頁數 77頁
口試委員 指導教授-簡素芳
委員-王啟銘
委員-張可中
中文關鍵字 α-半乳糖水解酵素  基因表現  表現質體  蛋白質純化  基因重組 
英文關鍵字 gene expression  Pichia pastoris  clones  integrate  oligosaccharide  transformants  enzyme activity 
學科別分類 學科別自然科學化學
中文摘要 α-半乳糖水解酵素(EC.3.2.1.22)能水解B型紅血球表面抗
原的α-半乳糖基,使B型紅血球可以轉換成O型紅血球。為了要
大量表現α-半乳糖水解酵素做紅血球轉型以利輸血之用,我們將
稻米的α-半乳糖水解酵素基因分別重組到表現質體pPICZαA及
pPIC9K上。並分別轉殖到酵母菌GS115及SMD1168內,目前挑選出來
表現最多的菌株為pPIC9K-αgal in SMD1168。目前可以表現在細胞內
每公升10毫克的α-半乳糖水解酵素;而細胞外的酵素每公升為1毫克。
分泌在細胞外的蛋白質可以省去很多純化過程,我們目前試著再改變培養
條件以增加產率。包括使用YNB-最簡單的培養基,加必要胺基酸及
乳糖;或增加供氧量等方式。發酵槽的培養可得到約10倍的基因表現
,我們接著應該一試。將來也可能試用昆蟲細胞、植物細胞及哺乳類
動物細胞,以達到大量表現酵素的目的。
英文摘要 α-galactosidase(EC. 3.2.1.22) is able to cleave the
terminal α-galactose from surface oligosaccharide chain of
B red blood cells, thus B type RBC can be converted into O type RBC.
In order to gain large quantity of enzyme for this purpose,
we tried to clone the rice α-galactosidase gene into
each of the pPICZαA and pPIC9K vector(Invitrogen®),
and to expression the enzyme in both GS115 and SMD1168 strain.
The transformants from pPIC9K/SMD1168 was obtained.
Expression the transformants in flask culture,
most enzyme activity was found intracellularly (10 mg
per liter culture);while there is about 1 mg per liter
culture of the enzyme activity was secreted in the media.
The secreted enzyme would be easier for further media
purification. From SDS-PAGE, the secreted enzyme revealed
as a major band. We have to try some other culture conditions to improve this results.
論文目次 目錄:
誌謝
中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
圖表目錄 Ⅴ
索引 Ⅶ
一、緒論
1.α-半乳糖水解酵素(α-galactosidase)的簡介 1
2. α-半乳糖水解酵素的應用 1
3.酵母菌(Pichia pastoris)蛋白質表現系統簡介 6
4.使用嗜甲基酵母菌的優點 10
5.研究目標 12
二、實驗材料
1.菌種與質體 13
2.培養基 13
3.酵素與受質 14
4.儀器 16

三、實驗方法
1.培養基配製 17
2.酵母菌表現質體及寄主的選擇 22
3.製備α-半乳糖水解酵素基因 27
4.製備表現質體 31
5.表現質體電導轉型到酵母菌 37
6.測試培養條件 47
7.用SDS-PAGE分析表現的酵素 50
8.紅血球轉型實驗 53
四、實驗結果與討論
1.製備α-半乳糖水解酵素基因 54
2.製備表現質體 56
3.表現質體電導轉型到酵母菌 59
4.測試培養條件 62
5.用SDS-PAGE分析表現的酵素 70
6.紅血球轉型實驗 71
五、結論與未來展望 72
六、參考資料 75


圖表目錄:
圖1. 血型抗原糖鏈結構 3
圖2. GL-3在人體的正常分解過程 5
圖3. Pichia pastoris的AOX 1 promoter 7
圖4. Gene insertion at AOX 1,gene replacement at AOX1。 9
圖5. Gene multi-integration 8
圖6.動物細胞與酵母菌表現的糖鏈比較 11
圖7. Zeocin結構 14
圖8. G418結構 15
圖9. pPICZαA and pPIC9K map 24
圖10-a. pPICZαA multiple cloning site 25
圖10-b. pPIC9K multiple cloning site 26
圖11. PCR洋菜電泳 55
圖12. 回收流程 30
圖13.表現質體gene cloning 31
圖14.水解pPICZαA-αgal電泳 57
圖15.水解pPIC9K-αgal電泳 57
圖16.菌種劃盤在α-X-gal plate反應結果 60
圖17.multicopy 洋菜電泳 61
圖18. MeOH對酵母菌培養的菌數和活性表現的影響 62
圖19. SDS-PAGE 分析誘導後培養基結果 70
圖20. 血球凝聚實驗結果 71
圖21: 2 copies gene vector製作 74

表1. 血液中血球跟抗體的凝聚現象 4
表2.Insert基因定序與稻米α-半乳糖水解酵素基因比對 59
表3.不同溫度的誘導結果 64
表4.不同菌數的誘導結果 66
表5.不同培養基的誘導結果 68
表6.不同菌種的誘導結果 69
表7.使用Pichia pastoris表現的酵素 72






索引:
中文 ;英文 頁數
α-半乳糖水解酵素;α-galactosidase 1
血凝現象 ;hemagglutination 3
法布瑞氏症 ;Fabry disease 4
嗜甲基酵母菌 ;methylotrophic yeast 6
整合 ;integrate(integration) 7
包覆體 ;inclution body 12
再摺疊 ;refolding 12
轉殖 ;transform 32
勝任細胞 ;competent cell 32
培養 ;incubate 32
熱休克 ;heat-shock 32
篩選 ;select 33
質體分離 ;plasmid DNA isolation 33
洗提 ;elute 33
電導轉型 ;electroporation 39
誘導 ;induce 44
轉型株 ;transformant 45

轉殖(實驗方法) 32
質體分離(實驗方法) 33
電導轉型(實驗方法) 39
分離酵母菌染色體DNA(實驗方法) 45


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