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研究生:林淑滿
研究生(外文):Lin Shu Man
論文名稱:抑菌素於大腸桿菌之表現與抗菌功能分析
論文名稱(外文):E. coli Expression and Antimicrobial Activity Study of Indolicidin
指導教授:毛嘉洪
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:英文
論文頁數:56
中文關鍵詞:抑菌素抗菌蛋白乳糖操縱子
外文關鍵詞:Indolocidinantimicrobial peptideCathelicidinpET32a(+)BL21 (DE3)
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抑菌素(indolicidin)是一種存於牛隻嗜中性白血球(neutrophil)顆粒中的抗菌蛋白(antimicrobial peptide),於非特異性自然免疫(innate immunity)中佔有重要角色,本研究藉由大腸桿菌(E. coli)宿主表現具有抗菌功能的indolicidin融合蛋白,期望能應用於對抗生素產生抗藥性之微生物感染。本實驗選用乳糖操縱子(lac operon)誘導表現載體pET32a(+),與合成的indolicidin基因片段連接後得到pET32a(+)indolicidin,將pET32a(+)indolicidin轉形入BL21 (ED3)大腸桿菌宿主(E. coli)表現出indolicidin融合蛋白。由組胺酸親和樹脂純化後,得到純化的indolicidin融合蛋白。經由enterokinase切割後,以E. coli (ATCC 25922)、S. aureus (ATCC 25923)及八株Sal. cholerasuis 菌株測其抗菌活性,結果以大腸桿菌表現產生的indolicidin並不具有抗菌活性,如何產生具抗菌功能的蛋白仍有待進一步的探討。

Indolicidin, an antimicrobial peptide that is found in the cytoplasmic granules of bovine neutrophil, plays an important role in innate immunity. The present study used Escherichia coli as a host to express indolicidin fusion protein and tested it for antimicrobial activity. It is hoped that the indolicidin produced by the E. coli could be used to control the microbial infection and might also replace the antibiotics that had been rendered ineffective by drug-resistant bacteria. The pET32a(+) vector with lac operon was used to induce and express the fusion protein. The pET32a(+)indolicidin, obtained by ligation of pET32a(+) and the synthesized indolicidin gene fragment, was transformed into an E. coli host BL21 (DE3), and a high level of indolicidin fusion proteins was expressed. Following purification by histidine affinity resin, purified indolicidin fusion protein was obtained. After enterokinase treatment, its antimicrobial activities were measured using E. coli (ATCC 25922), Staphylococcus aureus (ATCC 25923) and eight strains of Salmonella cholerasuis. However, no antimicrobial activity was observed for our expression protein. Thus further study is needed to produce an functional protein.

中文摘要…………………………………………………………….
英文摘要…………………………………………………………….
目次………………………………………………………………….
圖次………………………………………………………………….
表次………………………………………………………………….
壹、緒言…………………………………………………………….
貳、文獻探討…………………………………………………….
一、Cathelicidins 家族……………………………………..
二、Indolicidin名稱由來與歷史背景……………………..
三、Indolicidin的抗菌機制………………………………..
四、Indolicidin的抗菌功能分析…………………………..
五、Indolicidin的抗菌其他生物活性……………………..
參、材料與方法…………………………………………………
一、pET32a(+) indolicidin系列基因表現載體的構築與確認
二、pET32a(+)indolicidin系列基因表現載體的轉形與表現
三、抗菌蛋白活性試驗……………………………………….
肆、結果…………………………………………………………….
一、pET32a(+) indolicidin系列基因之表現載體構築與確認
二、pET32a(+)indolicidin系列基因表現載體的轉形與表現
三、抗菌蛋白活性試驗……………………………………….
伍、討論…………………………………………………………….
陸、參考文獻……………………………………………………….
附錄一:Indolicidin、I gene與I47基因序列(黑色網底為indolicidin基因序列)…………………………………..
附錄二:經定序確認pET32a(+)I47基因序列……………………
附錄三:pET32a(+)引導蛋白、I47蛋白、pET32a(+)I47融合蛋白序列…………………………………………………….
附錄四:經定序確認pET32a(+)indolicidin定序結果……………
附錄五:pET32a(+)引導蛋白、indolicidin蛋白、pET32a(+)indolicidin融合蛋白蛋白序列………………
附錄六:試驗使用之各項溶液配方……………………………….
附錄七:pET32a(+)載體的構築…………………………………….

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