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。

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.

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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