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研究生:楊翊梅
研究生(外文):Yi –Mei Yang
論文名稱:米麴菌之麩胺酸脫羧酶gad1及gad3基因選殖與表現
論文名稱(外文):Cloning and expression of the glutamate decarboxylase-encoding gene gad1 and gad3 from Aspergillus oryzae
指導教授:張耀南張耀南引用關係
指導教授(外文):Yaw-Nan Chang
學位類別:碩士
校院名稱:國立虎尾科技大學
系所名稱:光電與材料科技研究所
學門:工程學門
學類:電資工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:83
中文關鍵詞:米麴菌麩胺酸脫羧酶包涵體
外文關鍵詞:Aspergillus oryzaeglutamate decarboxylaseinclusion-body
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本研究由食品工業研究所生物資源保存中心所購得食用米麴菌Aspergillus oryzae BCRC 32288選殖出兩個不帶有intron的gad1與gad3基因,利用大腸桿菌大量表達麩胺酸脫羧酸酵素(glutamic acid decarboxylase; GAD EC 4.1.1.15),並以His-tag管柱純化且利用HPLC進行酵素定性分析。GAD可將麩胺酸(glutamate)轉化為γ-胺基丁酸(γ-aminobutyric acid; GABA)。gad1與gad3全長分別為1446 bp與1557 bp。將GAD1及GAD3蛋白核苷酸序列分析得知其分子量約為54 kDa及56kDa;與A. oryzae RIB40核苷酸序列比對,其相同性分別為99.6%。gad1基因選殖嵌入pET29表現載體以E.coli BLR(DE3)為宿主進行基因選殖與表現,其表達的蛋白質以包涵體的形式產生,活性為22.17 U/mg。gad3基因選殖分別嵌入pET29及pQE30表現載體,分別以E.coli BLR(DE3)及Novablue(DE3)為宿主進行基因選殖與表現,其表達的蛋白質以包涵體的形式產生,活性分別為42.4 U/mg及30.74 U/mg。測試gad基因於米麴菌本身的轉錄活性,分別以染色體DNA及mRNA進行PCR及RT-PCR皆可得到gad基因片段,證明在米麴菌中的gad基因並非為偽基因。再利用西方墨點法測試米麴菌蛋白合成,結果無法辨識到米麴菌所含的GAD蛋白。
The GAD-encoding gad1 and gad3 genes without gaps and introns from Aspergillus oryzae BCRC32288 obtained from Bioresouce Collection and Research Center, Food Industry Research & Development Institute, Hisingchu, Taiwan, were cloned and sequenced. The GAD (Glutamate acid or glutamate decarboxylase), which catalyzes α-decarboxylation reaction of glutamic acid (glutamate) to produce γ-aminobutyric acid (GABA), proteins were expressed in Escherichia coli and collected for purification by His-tag affinity column. The GAD activity was determined by the HPLC analysis method of GABA-OPA(o-phthalaldehyde) derivative. The two genes gad1 and gad3 from A. oryzae BCRC32288 consist of 1446 and 1557 nucleotides, respectively, and the nucleotide identities of gad1 and gad3 with those of A. oryzae RIB40 were 99.6%, respectively, while the GAD1 and GAD3 proteins expressed in E. coli have around 54kDa and 56kDa, respectively. When the gene gad1 was cloned into pET29 and over-expressed in E. coli BLR, the inclusion-body GAD1 activity was 22.17U/mg. When the gad3 was cloned into pET29 and over-expressed in E. coli BLR, and cloned into pQE30 and over-expressed in E. coli Novablue, the inclusion-body GAD3 activities were 42.40U/mg and 30.74U/mg for E. coli BLR and Novablue expression, respectively. The gene gad was obtained from chromosome DNA and mRNA of A. oryzae by using PCR and RT-PCR, respectively, in order to test the transcriptional activity. The result shows that the gene gad is not a pseudogene. However, GAD protein would not be identified by the western blot method.
中文摘要..i
Abstract..ii
誌謝......iii
表目錄....viii
圖目錄....ix
一、前言..1
二、文獻整理......2
2.1米麴菌簡介.....2
2.1.1 米麴之分類及生長型態..2
2.1.2 米麴菌培養方式........3
2.1.3 米麴菌之應用 ..........4
2.1.4 米麴菌之酵素..........5
2.1.5 米麴菌之基因 ..........9
2.2 γ-胺基丁酸簡介.........10
2.2.1 Glutamate與GABA之關係 .11
2.2.2 GABA定量:..12
2.2.3 GABA合成的調節........13
2.3 Glutamate decarboxylase .13
2.3.1 Calmodulin (CaM)-binding domain......15
2.3.2 pyridoxal 5’-phosphate (PLP)....15
2.4糖尿病與GABA....16
2.5專利檢索........17
三、材料與方法.....19
3.1實驗條件........19
3.1.1 實驗菌種及培養條件....19
3.1.2 緩衝液及溶劑.19
3.2染色體DNA抽取...20
3.3抽取total RNA...20
3.4 cDNA資料庫之建構........21
3.5聚合酶鏈鎖反應基因選殖法 (PCR cloning)....21
3.5.1 引子的設計...21
3.5.2 PCR ..22
3.6 DNA片段之回收..22
3.7限制酶切割......23
3.8 DNA連接反應....23
3.9轉形作用─電導法.....23
3.9.1 勝任細胞的製作....24
3.9.2 電穿孔.......24
3.9.3 轉形後重組質體之篩選確認......24
3.10米麴菌粗蛋白之萃取.....25
3.11 GAD酵素之純化 .........25
3.11.1 HisTrap FF管柱純化..25
3.11.2 以pQE30為表現載體之蛋白表現與純化....26
3.11.3 以pET29為表現載體之蛋白表現與純化....27
3.12 GAD酵素濃度之測定.....28
3.13 GAD酵素之定性 ........28
3.14多株抗體之製作 ........29
3.14.1 血清的製備 ........30
3.14.2西方墨點法(western blot)....30
四、結果 ..32
4.1米麴菌GAD酵素之基因選殖...32
4.1.1 Aspergillus oryzae RIB40 gad基因之序列分析....32
4.1.2 Aspergillus oryzae BCRC 32288 gad基因之PCR DNA片段電泳分析............32
4.1.3重組質體之構築.........33
4.2選殖之gad1及gad3序列分析 .34
4.3米麴菌gad1及gad3基因表現分析....35
4.3.1 選殖之GAD蛋白表現分析 ........35
4.3.2 利用RT-PCR測定gad基因轉錄活性.37
4.4 GAD酵素活性之分析......38
4.4.1偵測波長之設定........38
4.4.2酵素比活性之測量......38
五、討論...40
附錄.....51
Extended Abstract.80
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