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研究生：	游舒琇 Shu-Hsiu Yu
論文名稱：	人類海藻糖水解酶重組蛋白之表達 Expression of the Recombinant Human Trehalase
指導教授：	李冠群 Lee, Guan-Chiun
學位類別：	碩士 Master
系所名稱：	生命科學系 Department of Life Science
論文出版年：	2014
畢業學年度：	102
語文別：	中文
論文頁數：	102
中文關鍵詞：	海藻糖、人類海藻糖水解酶、大腸桿菌、畢赤酵母菌、桿狀病毒、sf9 昆蟲細胞
英文關鍵詞：	trehalose, human trehalase, E. coli, Pichia pastoris, baculovirus, sf9 cell
論文種類：	學術論文
相關次數：	點閱：71 下載：3
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海藻糖 (trehalose) 是一種非還原性的雙糖，由兩個葡萄糖分子以 α,α-1,1- 糖苷鏈結形成，存在於多種生物體中，除了可以做為生物能量來源，亦有穩定蛋白質與細胞膜等重要生理功能。海藻糖水解酶 α,α-Trehalase (TreH) (EC 3.2.1.28)，可將海藻糖水解成兩個葡萄糖，廣泛的存在於微生物與動植物中。人體不同組織中亦含有 TreH，例如小腸與腎臟絨毛膜的 TreH 負責海藻糖的水解以利吸收，另外，血漿中亦含有TreH，有研究顯示血漿中具有高 TreH 活性的人較容易罹患糖尿病。然而，目前為止對於人類 TreH 的生化特性、功能與結構的研究非常稀少。本研究將選殖的兩種人類海藻糖水解酶 isoform1 (hTreH1) 和 isoform2 (hTreH2) 的基因，利用三種蛋白表達系統，包括原核生物的大腸桿菌 (Escherichia coli) 表達系統、真核生物的畢赤酵母菌 (Pichia pastoris) 表達系統和昆蟲桿狀病毒表達系統 (baculovirus expressing vector system)，以便生產具功能性的重組酵素。其中，兩種 hTreHs 在大腸桿菌表達都形成不可溶的內涵體，以隨機突變技術篩選 80 個突變株，並未獲得可表達水溶性酵素之突變株；在畢赤酵母菌表達出的胞外重組酵素量極微，在培養液中無法偵測到 TreH 活性；而在昆蟲桿狀病毒表達系統中有表達出重組酵素，但是仍然形成不可溶的蛋白沉澱。部分可溶的重組蛋白可以非變性的蛋白萃取條件萃出，然而這些萃出的重組蛋白還是不具活性。本研究所採用的方法中沒有表達出具有活性的重組蛋白，為了得到可溶且具有活性的重組蛋白，需要再進一步做更深入的研究。

Trehalose is a non-reducing disaccharide formed by two glucose molecules linked by a 1α-1α glycosidic bond. It occurs in a wide range of bacteria, fungi, insects, invertebrates and plants. Apart from being as energy source, trehalose has many important physiological functions such as stabilizing proteins and cell membranes. α,α-Trehalase (EC 3.2.1.28) (TreH), which hydrolyzes α,α-trehalose to two glucose molecules, is widespread in nature and found in various human tissues as well as in plasma. It has been reported that people with high plasma trehalase activity are more prone to develop diabetes mellitus than people with low enzyme activity. However, the biochemical property and structure of human TreH have not been determined. In order to produce functional recombinant human trehalases (hTreH), hTreH1 and hTreH2 were expressed in various protein expression systems including prokaryotic E. coli, eukaryotic yeast Pichia pastoris and insect cell systems. In the E. coli expression systems, hTreHs were expressed as inclusion body. Random mutagenesis and screening for improving recombinant protein solubility were performed and no improved mutants were screened among eighty transformants. In Pichia pastoris, the expression level of secretory hTreHs were very low and no activity were detected in the culture medium. In insect cell systems, hTreHs can be expressed, but appeared as insoluble form. Some soluble recombinant hTreHs were extracted under non-denaturing conditions, however in biologically inactive forms. Further investigations are needed to obtain the soluble biologically active forms of recombinant hTreHs.

表次    VII
圖次    VIII
附錄    XI
摘要    XII
Abstract    XIII
縮寫名詞對照    XIV
壹、緒論    1
一、海藻糖的簡介    1
二、海藻糖的性質    1
三、海藻糖的應用    2
四、海藻糖水解酶 (Trehalase, TreH) 的介紹    4
五、人類海藻糖水解酶 (human trehalase, hTreH)    5
六、海藻糖水解酶抑制劑    6
七、蛋白表達系統簡介    7
(一)、大腸桿菌表達系統 (Escherichia coli)    7
(二)、畢赤酵母表達系統 (Pichia pastoris)    7
(三)、昆蟲桿狀病毒表達載體系統 (baculovirus expressing vector system； BEVS)    7
貳、研究目的    9
參、材料與方法    10
一、人類海藻糖酶基因的取得    10
(一)、 hTreH1 (hTreH iosform1 gene)    10
(二)、 hTreH2 (hTreH iosform2 gene)    10
二、蛋白表達宿主細胞及培養液    10
(一)、大腸桿菌    10
(二)、畢赤酵母    10
(三)、昆蟲細胞    10
(四)、培養液    11
三、基因載體    11
(一)、大腸桿菌表達系統質體    11
(二)、畢赤酵母表達系統質體    12
(三)、昆蟲桿狀病毒表達載體系統質體    12
四、質體 DNA 製備    13
五、 DNA 之定量與電泳    14
(一)、 DNA 定量    14
(二)、 DNA 電泳    14
六、各個表達系統-構築質體    14
(一)、大腸桿菌表達系統-構築質體    14
(二)、畢赤酵母表達系統-構築質體    15
(三)、昆蟲桿狀病毒表達載體系統-構築質體    16
七、 DNA 定序分析    19
八、 hTreHs 重組蛋白的表達    19
(一)、大腸桿菌表達系統    19
1.大腸桿菌的轉型作用    19
2. hTreH1 的表達    19
(二)、畢赤酵母表達系統    20
1. P. pastoris 的轉型作用    20
2. hTreHs 的表達    21
(三)、昆蟲桿狀病毒表達載體系統    22
1.昆蟲桿狀病毒製備    22
2. hTreHs 的表達    24
九、以非變性萃取法獲得可溶之 hTreHs    25
十、 hTreHs 純化分析    26
(一)、固定化金屬親和性層析法純化蛋白質    26
(二)、高效能 96 孔盤型式蛋白質的純化與透析    26
1.高效能 96 孔盤型式蛋白質的純化    27
2.高效能 96 孔盤型式蛋白質去鹽    27
3. PD MultiTrap G-25 回收再利用    27
十一、 hTreHs 蛋白質分析    28
(一)、蛋白質電泳    28
1.蛋白質電泳試劑：    28
2. SDS-聚丙醯胺膠體溶液調配法：    28
3.膠體的製備：    28
4. 5X SDS sample buffer製備與電泳條件：    29
5.電泳膠體的染色和退染：    29
(二)、蛋白質的定量    29
十二、 hTreHs 酵素活性分析    30
(一)、 HPLC (High-performance liquid chromatography)    30
(二)、 glucose assay    31
十三、 hTreH1 酵素受質專一性與 Validamycin A 的抑制實驗    32
(一)、 hTreH1 酵素受質專一性：    32
(二)、 hTreH1 與 Validamycin A 的反應：    32
肆、研究結果    33
一、 hTreHs 基因的分析    33
二、大腸桿菌表達系統    33
(一)、 hTreH1 基因重組質體之建構    33
(二)、 hTreH1 蛋白質的活性分析    33
三、畢赤酵母菌表達系統    33
(一)、 hTreHs 基因重組質體之建構    33
1. pGAPZαC    33
2. pPIC9K    34
(二)、 hTreHs 基因的表達    34
1. pGAPZαC 的表達    34
2. pPIC9K 表達    35
(三)、 hTreHs 蛋白質的活性分析    35
三、昆蟲桿狀病毒表達載體系統    36
(一)、 hTreHs 基因重組質體之建構    36
1. Bacvector3000 - pTriEx7-EK/LIC    36
2. BaculoDirect - pENTR/SD/D-TOPO    36
(二)、 hTreHs 的表達    36
1. Bacvector3000    36
2. BaculoDirect    37
(三)、 hTreHs 蛋白的活性分析    38
1. Bacvector3000    38
2. BaculoDirect    38
四、 hTreH1 酵素受質專一性與 Validamycin A 的抑制實驗    39
(一)、 hTreH1 酵素受質專一性    39
(二)、 hTreH1 與 Validamycin A 的反應    39
伍、討論    40
一、 hTreH1 在大腸桿菌的表達    40
二、 hTreHs 在畢赤酵母的表達    40
三、 hTreHs 在昆蟲桿狀病毒的表達    41
四、 hTreH1 酵素受質專一性與 Validamycin A 的抑制實驗    43
陸、參考文獻    45
                                

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