本研究目的是針對Bacillus subtilis TKU007納豆激酶(nattokinase)，利用比對、選殖的方式去找出全長基因且研究其中與穩定性相關的基因序列。將TKU007納豆激酶基因轉殖入其他異源表現宿主中偵測其表現活性。首先利用聚合酶連鎖反應進行基因的放大，並轉型入pGEM-T easy vecetor做TA-cloning，將其質體DNA定序與B. subtilis 比對確認序列的正確性。發現TKU007納豆激酶基因有幾個胺基酸與一般B. subtilis的納豆激酶基因不同，顯示出這TKU007與B. subtilis之間這幾個胺基酸之差異，可能因此造成物化特性或是其他酵素活性表現的差異。也試著將TKU007所分泌的納豆激酶表現在異源的酵母菌細胞中，藉此試驗是否在較複雜的真核生物體中也同樣能有其蛋白質活性的表現。將序列重組於pYES2 表現載體，其載體中含有半乳糖的promoter，可用半乳糖去誘導，在啤酒酵母菌(BJ2168)中去試著表現。也將序列重組於pET-26大腸桿菌的表現載體在原核大腸桿菌BL21(DE3)中，以乳糖誘導表現。

This research was first to find the complete gene sequence of nattokinase of Bacillus subtilis TKU007. We are also interested in the correlation of the gene sequence and enzyme stability. The B. .subtilis TKU007’s gene was transformed into heterologous hosts to over-express the protein for activity detection. First, the gene was amplified using the polymerization chain-reaction and ligated to pGEM-T easy vecetor by TA-cloning. Sequence comparison between TKU007 and common B.. subtilis discovered that the TKU007 nattokinase have 2 amino acid substitutions.These differences perhaps create the different enzyme properties in the TKU 007 nattokinase. 
  We first tired to over-express TKU007 nattokinase heterologously in the Saccharomyces cerevisiae BJ2168 cells using pYES2 expression vector, containing the galactose promoter which was thus induced by galactose. We also over-expressed this nattokinase in E. coli BL21(DE3) expression using pET-26 vector, which was induced by lactose.

目錄
中文摘要----------------------------------------------------Ⅰ
英文摘要----------------------------------------------------Ⅱ
目錄--------------------------------------------------------Ⅲ
第一章 緒論--------------------------------------------------1
1-1枯草桿菌 TKU007與納豆激酶-------------------------------1
1-2 簡併引子-------------------------------------------------2
第二章 材料與方法--------------------------------------------3
第一節  TKU007納豆激酶之選殖-------------------------------3
1-1	養小量TKU007做菌種保存--------------------------------3
 1-1-1菌種保存-----------------------------------------------3
1-2  聚合酶連鎖反應
 1-2-1 設計引子之要素----------------------------------------5
  1-2-1.A   搜尋比對相關枯草桿菌的序列-----------------------6
 1-2-2 模版製備 -------------------------------------------- 12
 1-2-3 聚合酶連鎖反應--------------------------------------- 13
 1-2-4 鑑定PCR產物----------------------------------------- 15
1-4  pGEM-T easy載體接合轉型至大腸桿菌勝任細胞DH5α---------18
 1-4-1 補鹼基A----------------------------------------------18
 1-4-2 接合反應--------------------------------------------- 19
 1-4-3 用氯化鈣轉型法送入大腸桿菌養大量與藍白篩選-----------21
1-5  抽取質體DNA-------------------------------------------22
1-6  限制酶切割反應----------------------------------------- 24
第二節  在酵母菌中TKU007分泌納豆激酶的誘導表現------------ 25
2-1 引子設計------------------------------------------------ 26
2-2  納豆激酶連接入pYES 2並轉型送入酵母菌BJ2168中---------26
 2-2-1酵母菌轉型(Yeast Transform)---------------------------- 27
2-3  在酵母菌BJ2168中誘導表現納豆激酶---------------------- 28
2-4	利用SDS電泳分析蛋白質表現-----------------------------29
第三章 結果與討論------------------------------------------- 34
第一節  TKU007分泌的納豆激酶之選殖------------------------ 34
1-1 簡併引子進行PCR確認片段長度----------------------------34
1-2 相似序列段引子------------------------------------------ 37
第二節  在酵母菌及大腸桿菌中TKU007分泌納豆激酶的誘導表現---43
2-1 接上表現載體pYES2轉型至BJ2168--------------------------43
2-2 在BJ2168中誘導表現結果----------------------------------44
2-3 在大腸桿菌BL21(DE3)中TKU007分泌納豆激酶誘導表現-------46
第四章 結論-------------------------------------------------47 
參考文獻  49

圖表目錄
表一、實驗過程所用的載體(vector)、切位及宿主細胞-----------------3
表二、配製PCR 反應物------------------------------------------14
表三、溫度變化-------------------------------------------------15
表四、PCR 產物補A--------------------------------------------19
表五、接合反應（ligation）配製反應溶液--------------------------20
表六、限制酶切位建立之反應組成--------------------------------25
表七、鑄膠組成-------------------------------------------------31
表八、配製4％焦集膠體溶液-------------------------------------32
表九、pGEM Easy vector的最佳效能公式--------------------------47
圖a、BSUB Physical Map-----------------------------------------10
圖1-1.a、溫度48度 2%瓊脂膠電泳圖-----------------------------34
圖1-1.b、溫度為45度，2%瓊脂膠電泳圖--------------------------35
圖1-1.c、加入DMSO 溫度為53度，2 %瓊脂膠電泳圖---------------35
圖1-1.d、溫度為55度，2 %瓊脂膠電泳圖--------------------------36
圖1-1.e、溫度為53度，循環30次，2 %瓊脂膠電泳圖----------------36
圖1-2、溫度是55度，循環40次，2%瓊脂膠電泳圖-----------------38
圖1-3、N1N4轉型至pGEM-T easy載體後E coRI切位確認------------38
圖1-4、1NF和1NB進行PCR後的結果，2%瓊脂膠電泳圖------------39
圖1-5、4NF和4NB進行PCR後的結果，2 %瓊脂膠電泳圖-----------39
圖1-6、Bacillus subtilis與TKU007 納豆激酶鹼基序列比對-----------42
圖1-7、Bacillus Subtilis的序列與TKU007胺基酸序列比對-------------42
圖2-1、基因接上pYES2以限制酶SacI和E coRI切位----------------44
圖2-2 、在BJ2168中用galactose誘導跑SDS-PAGE的結果-----------45
圖2-3 :在BL21中用lactose誘導跑SDS-PAGE的結果----------------46

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