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研究生:許淑芳
研究生(外文):Joyce S. F. Hsu
論文名稱:愛玉子瘦果幾丁質水解酶基因之選殖與表現
論文名稱(外文):Cloning and Expression of a Chitinase from Jelly Fig (Ficus awkeotsang Makino) Achenes
指導教授:曾志正曾志正引用關係
指導教授(外文):Jason T. C. Tzen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
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
論文頁數:71
中文關鍵詞:幾丁質水解酶愛玉子瘦果
外文關鍵詞:chitinasejelly fig achenes
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愛玉子 (Ficus awkeotasang Makino) 是一種常綠的木質藤本植物,藉氣根攀附於林木枝幹或岩石上生長,為台灣特有植物。由搓洗愛玉子瘦果 (achenes) 所得之愛玉凍則長久為溽暑中消暑之飲品。愛玉子雖為本省特產,但並非生活必需品,因此前人研究甚少。愛玉凍中含有愛玉子瘦果水溶性蛋白質,其中含量最豐富者已被鑑定出是一種酸性果膠甲基酯酶 (pectin methylesterase) ,其功能與愛玉子的凝膠相關。本研究針對含量居第二,分子量約30 kDa的蛋白質,進行其對應基因的選殖並探討其生理功能。首先將蛋白質經SDS-PAGE純化收集,進行N端胺基酸定序 (amino acid N-terminal sequencing) 並製備抗體。利用製備好之抗體進行免疫篩選基因選殖(immunoscreen cloning) ,並根據N端胺基酸定序所得胺基酸序列設計一條退化引子 (degenerate primer) ,搭配選殖載體 (cloning vector) 上的專一性引子 (specific primer) ,進行聚合酶連鎖反應基因選殖 (PCR cloning) 。由免疫篩選基因選殖得到一cDNA序列,此cDNA序列經由大腸桿菌 (E. coli) 蛋白質表現系統 (protein expression system) 進行大量表現所得之蛋白質分子量為15.8 kDa且無法被先前製備之抗體辨識,顯示此cDNA並非愛玉子瘦果30 kDa蛋白質的對應基因。此cDNA轉譯出之胺基酸序列在基因庫 (GenBank) 中與已發表之蛋白質序列相較,推測其為一種子成熟過程中誘導表現之蛋白質 (ripening-induced protein) 。而由聚合酶連鎖反應基因選殖得到的cDNA序列經由大腸桿菌大量表現所得之蛋白質分子量為29.6 kDa且可被先前製備之抗體辨識。在基因庫 (GenBank) 中與已發表之蛋白質序列相較,可知此cDNA轉譯出一幾丁質水解酶 (chitinase) 。此幾丁質水解酶之酵素活性可由愛玉凍中純化所得的蛋白質上得到證實。而大腸桿菌所表現之幾丁質水解酶重組蛋白均形成包涵體 (inclusion bodies) 。此包涵體經9 M尿素回溶,以透析法回復蛋白結構,並進行酵素活性檢測,結果顯示經由大腸桿菌表現之愛玉子瘦果幾丁質水解酶重組蛋白並不具有幾丁質水解酶活性。

Jelly fig (Ficus awkeotasang Makino) is a unique woody vine growing in the mountain areas of Taiwan. Water extract of jelly fig achenes has been extensively utilized in preparation of a unique jelly curd for making a popular drink. The major protein extracted from jelly curd has been identified as an acidic pectin methylesterase responsible for the gelation. In this study, the second abundant protein 30 kDa extracted from the curd was subjected to N-terminal sequencing and antibody preparation. A cDNA sequence was obtained by immunoscreen cloning with antibody recognition. The size of its over-expressed protein in E. coli was not match with 30 kDa. And the E. coli over-expressed protein can’t be recognized by anti-30 kDa antibody. Sequence comparison with published protein sequences in GenBank reveals that the clone encodes a ripening-induced protein. A degenerate primer was designed according to the partial amino acid sequence. A cDNA sequence was obtained by PCR cloning using the degenerate primer and a specific primer of the cloning vector. The cDNA clone was subsequently confirmed via immunological recognition of its over-expressed protein in E. coli. Sequence comparison with published protein sequences in GenBank reveals that the clone encodes a chitinase. Chitinase activity was detected in the 30 kDa protein purified from jelly curd. However the E. coli over-expressed recombinant chitinase was lack of enzymatic activity.

第一章 前言…………………………………………………1
第二章 文獻回顧
一、幾丁質水解酶簡介………………………………………4
二、植物幾丁質水解酶……………………………………………6
三、動物及微生物幾丁質水解酶…………………………………7
四、幾丁質水解酶的應用………………………………………8
第三章 材料方法
第一節 實驗材料…………………………………………………10
第二節 實驗方法…………………………………………………10
一、愛玉子瘦果胞外蛋白質之萃取………………………………10
二、SDS-聚丙烯醯胺膠體電泳…………………………………11
三、多株抗體之製備………………………………………………13
四、西方墨點法之分析……………………………………………14
五、免疫篩選基因選殖法…………………………………………16
六、聚合酶連鎖反應基因選殖法…………………………………20
七、愛玉子瘦果幾丁質水解酶之酵素活性測定…………………25
八、愛玉子瘦果幾丁質水解酶基因於大腸桿菌中表現…………25
九、愛玉子瘦果幾丁質水解酶重組蛋白之活性測定……………26
第四章 結果
一、愛玉子瘦果胞外蛋白之萃取及30 kDa蛋白質之純化………28
二、 30 kDa蛋白質的抗體製備及西方墨點法進行免疫檢測…28
三、 30 kDa蛋白質對應基因之選殖………………………………29
四、愛玉子瘦果幾丁質水解酶之酵素活性測定…………………32
五、利用大腸桿菌誘導表現愛玉子瘦果幾丁質水解酶…………32
六、愛玉子瘦果幾丁質水解酶重組蛋白之活性測定………………33
第五章 討論…………………………………………………………34
第六章 結論…………………………………………………………40
第七章 參考文獻……………………………………………………41
圖………………………………………………………………………48
圖目錄
圖一、愛玉子植株……………………………………………………..48
圖二、愛玉子雌隱花果…………………………………………………49
圖三、愛玉子瘦果………………………………………………………50
圖四、幾丁質(1)和纖維素(2)之結構…………………………………51
圖五、族群18(1)及族群19(2)幾丁質水解酶胺基酸序列保留區…..52
圖六、巴西橡膠樹幾丁質水解酶三度空間立體結構…………………53
圖七、植物利用幾丁質水解酶防禦真菌之模式圖……………………54
圖八、聚合酶連鎖反應基因選殖法選殖基因之策略…………………55
圖九、愛玉子瘦果胞外蛋白質粗萃取之蛋白質電泳圖………………56
圖十、利用割膠方式純化30 kDa蛋白質之蛋白質電泳圖及西方墨點
法進行免疫檢測之結果………………………………………..57
圖十一、第一次免疫基因篩選(1)及第三次免疫基因篩選(2)之結果..58
圖十二、Ripening-induced protein cDNA核酸序列及轉譯之胺基酸序
列……………………………………………………………..59
圖十三、利用IPTG誘導ripening-induced protein於大腸桿菌中大量表
現之蛋白質電泳圖…………………………………………..60
圖十四、Ripening-induced protein於大腸桿菌中大量表現之蛋白質電泳
圖及西方墨點法進行免疫檢測之結果……………………..61
圖十五、胰凝乳蛋白酶及胰蛋白酶水解30 kDa蛋白質之電泳圖…62
圖十六、幾丁質水解酶胺基酸序列比對及5´端退化引子的設計…63
圖十七、愛玉子瘦果幾丁質水解酶cDNA之核酸序列及轉譯之胺幾酸
序列……………………………………………………………64
圖十八、愛玉子瘦果幾丁質水解酶胺基酸序列與rubber tree、pokeweed
比對之結果及其二級結構預測圖……….…………………...65
圖十九、愛玉子瘦果幾丁質水解酶三度空間立體結構預測圖………66
圖二十、利用IPTG誘導愛玉子瘦果幾丁質水解酶於大腸桿菌中大量
表現之蛋白質電泳圖及西方墨點法進行免疫檢測之結果..67
圖二十一、愛玉子瘦果幾丁質水解酶之SDS-PAGE酵素活性染色圖
……………………………………………………………68
圖二十二、愛玉子瘦果幾丁質水解酶在大腸桿菌BL21中表現形成包
涵體之蛋白質電泳圖……………………………………..69
圖二十三、愛玉子瘦果幾丁質水解酶在大腸桿菌AD494中表現形成
包涵體之蛋白質電泳圖及西方墨點法進行免疫檢測之結
果…………………………………………………………..70
圖二十四、大腸桿菌表現之幾丁質水解酶重組蛋白之SDS-PAGE酵
素活性染色圖……………………………………………..71

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