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研究生:賴宗徽
論文名稱:洛德乳酸桿菌脂酸輔酵素A(Acyl-CoA)水解酵素之選殖、定序、表現與功能分析
論文名稱(外文):Cloning, sequencing, expression and enzymatic activity assay of an Acyl-CoA hydrolase from lactobacillus reuteri
指導教授:張登欽張登欽引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫微生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:洛德乳酸桿菌
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摘 要
乳酸菌被運用在食品工業上,已經有很長的歷史,目前更朝口服疫苗的方向發展,期能經由刺激消化道黏膜免疫以保護動物。本實驗室曾以shot-gun cloning與澱粉酵素活性分析的方式,得到6段能將產物分泌至培養液中的DNA片段。為瞭解這些序列於洛德乳酸桿菌中所扮演的角色,本實驗室針對其中兩段以chromosomal walking與PCR技術將其下游核酸序列增幅出來,並以自動定序的方式得到其序列。所得到的序列經與GenBank的data base比對,得知分別與Listeria innocua的Acyl-CoA水解酵素及Streptococcus pyogenes的RNA聚合酵素之次單元delta有37%及50%之相似度(similarity)。本實驗更進一步針對acyl-CoA 水解酵素之基因設計一對引子,以PCR的方式將其從染色體中增幅出來,並接入選殖載體pUC19的Sma I切位中。之後再將含有acyl-CoA水解酵素開讀框(open reading frame)的片段以限制酵素Nco I 與 Sac I切出並接入表現載體pET32之Nco I 與 Sac I切位中。利用IPTG誘導能使此表現系統表現融合蛋白。在以6xHis resin進行親和性純化(affinity purification)後,以SDS-PAGE分析,可觀察到一分子量約22 kDa的融合蛋白質。經由Palmitoyl-CoA水解的結果顯示,本次實驗建構的重組蛋白具有acyl-CoA水解酵素的活性。

目錄
第一章、 緒言………………………………………………………………………………………………………………………1
第二章 文獻探討……………………………………………………………………………………………………………………2
2.1 乳酸菌之簡介………………………………………………………………………………………………………2
2.2 乳酸菌在動物體中所扮演的角色…………………………………………………………………2
2.3 洛德乳酸桿菌在分子生物學方面之研究……………………………………………………2
2.3.1 代謝性酵素之研究………………………………………………………………………………………2
2.3.2 洛德乳酸桿菌質體之研究……………………………………………………………………………3
2.3.3 洛德乳酸桿菌與高膽固醇血症…………………………………………………………………3
2.4 利用洛德乳酸桿菌對抗病原菌………………………………………………………………………4
2.4.1 利用洛德乳酸桿菌,抑制幽門螺旋菌與醣脂受體之結合……………………4
2.4.2 利用乳酸菌作為活毒疫苗載體之研究……………………………………………………4
2.4.3 分泌性蛋白質…………………………………………………………………………………………………5
2.4.4 乳酸菌表現系統之開發………………………………………………………………………………5
2.5 探知一基因上下游序列之方法………………………………………………………………………6
2.5.1 利用基因庫來尋找………………………………………………………………………………………6
2.5.2 利用PCR的方式………………………………………………………………………………………6
2.5.3 TaKaRa所出品的LA PCR in vitro Cloning Kit……………………………7
2.6 Acyl-CoA hydrolase於生物體內所扮演的角色………………………………7
2.7 陽性菌脂肪酸代謝中acyl-CoA hydrolase活性分析法…………………8
第三章 材料與方法………………………………………………………………………………………………………………9
3.1 菌株………………………………………………………………………………………………………………………9
3.2 培養液…………………………………………………………………………………………………………………9
3.3 藥品………………………………………………………………………………………………………………………10
3.4 選殖洛德乳酸桿菌分泌性蛋白基因之策略………………………………………………10
3.4.1 洛德乳酸桿菌染色體之製備………………………………………………………………10
3.4.2 洛德乳酸桿菌染色體之限制酵素切割、agarose gel電泳分析、與毛細管轉漬………………………………………………………………………………………12
3.4.3 含有訊號序列之質體DNA製備………………………………………………………………13
3.4.4南方墨點法探針之製備………………………………………………………………………………14
3.4.5 探針DNA之標記……………………………………………………………………………………16
3.4.6 以南方轉漬法求出含有目標基因之限制酵素染色體切割片段的分子量大小…………………………………………………………………………………………………………16
3.5 將含有目標基因之DNA片段與選殖載體pUC19接合並用PCR將含有目標序列的核酸片段增幅出來……………………………………………………………17
3.5.1 選殖載體pUC19之製備、限制酵素切割、與去磷處理………………………18
3.5.2 洛德乳酸桿菌染色體之切割核酸片段純化與接入pUC19………………19
3.5.3 以聚合酵素連鎖反應增幅含有目標基因之片段…………………………………19
3.6 具訊號序列活性之核酸序列分析與比對……………………………………………………21
3.7 以PCR進行Acyl-CoA hydrolase基因之選殖…………………………………21
3.7.1 根據定序結果從基因的頭尾設計引子與PCR反應……………………………21
3.7.2 將PCR產物接入pUC 19之Sma I切位:………………………………22
3.7.3 轉型入大腸桿菌TG1勝任細胞:……………………………………………………………22
3.8 以pET系統表現與純化洛德乳酸桿菌之acyl-CoA hydrolase…23
3.8.1 表現載體pET28a之製備…………………………………………………………………………23
3.8.2 限制酵素切割………………………………………………………………………………………………23
3.8.3 送入勝任細胞大腸桿菌BL21中:…………………………………………………………24
3.8.4 菌株之篩選……………………………………………………………………………………………………24
3.8.5 Acyl-CoA hydrolase —His tag 融合蛋白之誘導表現…………24
3.9 純化後之acyl-CoA hydrolase酵素活性分析…………………………………26
3.10 本酵素對於碳數14 16與18之acyl-CoA活性之差異……………………27
3.11 Acyl-CoA hydrolase之Km與Vmax之計算……………………………………27
第四章、結果…………………………………………………………………………………………………………………………28
4.1 洛德乳酸桿菌染色體之抽取與限制酵素切割………………………………………28
4.2 本實驗室選殖出具訊號序列活性DNA序列,其在洛德乳酸桿菌染色體經各類限制酵素切割後的片段大小…………………………………………………………28
4.3 本實驗室選殖出具訊號序列活性DNA片段,其序列可能表現的基因28
4.4 將洛德乳酸桿菌acyl-CoA水解酵素選殖至表現載體pET32a並建立一能經誘導而大量表現並且純化此酵素的菌株……………………………………29
4.5 純化所得之acyl-CoA水解酵素證實確有酵素活性……………………………29
第五章、討論…………………………………………………………………………………………………………………………47
參考文獻………………………………………………………………………………………………………………………52
附錄………………………………………………………………………………………………………………………………………57

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