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研究生:吳哲彰
研究生(外文):Che-chang Wu
論文名稱:愛德華氏菌及其噬菌體培養分析
論文名稱(外文):Culture Analysis of Edwardsiella tarda and Bacteriophage
指導教授:吳聲祺
指導教授(外文):Sheng-chi Wu
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:生物技術系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:68
中文關鍵詞:噬菌體愛德華氏菌褐藻酸
外文關鍵詞:bacteriophageEdwardsiella tardaalginate
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現今對於致病性細菌疾病的防治,大多是以使用抗生素方式來給予治療,而這種治療方式雖然快速有效,卻也出現使菌株產生抗藥性的新問題。E.tarda,屬於兼性厭氧革蘭氏陰性短桿菌,是引起魚類疾病的病原菌中的一種,也有研究指出在豬隻疾病上發現E.tarda的蹤跡,而在豬隻感染E.tarda之後可能會導致豬隻肝臟潰瘍,進而產生更多疾病,如:肛門紅腫、腎臟壞死。而人類感染則會引起腸胃炎、腦膜炎等併發症。
本研究嘗試利用噬菌體溶裂愛德華氏菌(Edwardsiel-la tarda),以評估噬菌體在致病性細菌治療上的可行性和效果。在進行測試前,我們先探討大量生產噬菌體所需之條件。對培養細菌的LB培養基的pH值及培養溫度還有成分比例進行優化,在經由Design expert軟體的實驗設計及實驗之後,我們得到一組最佳培養條件,pH為6.5、培養溫度為38.62℃,培養基成分方面,則為Glucose 0.57(g/L)、Tryptone 18.70(g/L)及Yeast extract 6.68(g/L)。在病毒感染方面,我們在搖瓶培養下可得最大噬菌體生產量1.27×1010PFU/ml,而在醱酵槽放大培養噬菌體時,可得1.35×109PFU/ml的噬菌體量。噬菌體特性方面,其在pH3至pH3.5之間的耐受性非常低。接著我們利用包埋法將噬菌體包埋在褐藻膠顆粒中,並進行模擬胃腸道測試,結果噬菌體顆粒可以在pH2.4之酸性環境下維持活性,未來可做為治療方法。
Using antibiotics to treat pathogenic disease has many drawbacks such as inducing resistant strains. In this study, we used bacteriophage against to Edwardsiella tarda as a model system to develop an alternative strategy to prevent the bacteria infection.
At first, we used Design expert to optimize the cultural condition for E. tarda, and found the optimal pH, temperature, glucose addition, tryptone addition and yeast extract addition are 6.5, 38.62℃, 0.57 g/L, 18.7 g/L, 6.68 g/L, correspondingly. After infection with the bacteriophage, we recovered 1.27×1010 and 1.35×109 PFU/ml of bacteriophage from a flask and a fermentor, respectively.
The microencapsulated bacteriophage by alginate for oral delivery was also developed in this study. Free phage was found to beextremely sensitive to acidic environments and was serious decreased at pH3~3.5. In contrast, microencapsulated phage had a high viability at pH 2.4. So the encapsulated phage has the potential in disease treatment.
誌謝 i
中文摘要 ii
Abstract iii
表次 vi
圖次 vii
第一章 前言 1
第二章 文獻回顧 3
第一節 菌種背景及介紹 3
第二節 噬菌體的發現及特性 4
第三節 噬菌體治療的應用及優點 5
第四節 褐藻膠介紹及應用 5
第三章 材料與方法 7
第一節 藥品與儀器 7
第二節 培養基與藥品配置 9
第三節 菌種培養與保存 10
第四節 菌種培養條件最適化 12
第五節 菌株生長曲線測定 12
第六節 噬菌體固態培養 12
第七節 噬菌體液態培養及測試 15
第八節 噬菌體醱酵槽放大培養 15
第九節 噬菌體的濃縮純化 16
第十節 噬菌體酸鹼值耐受測試 16
第十一節 噬菌體包埋及顆粒的菌株溶裂能力 16
第十二節 包埋噬菌體顆粒在酸性條件下的存活率 20
第十三節 噬菌體顆粒在人工腸液下的體外釋放測試 20
第四章 實驗結果 21
第一節 菌株培養最佳條件 21
第二節 培養基修改前後菌株生長曲線比較 32
第三節 不同濃度噬菌體溶裂能力測試 35
第四節 噬菌體醱酵槽放大培養 43
第五節 噬菌體濃縮 46
第六節 酸鹼值對於噬菌體的影響 48
第七節 噬菌體顆粒的溶裂能力測試 50
第八節 噬菌體顆粒在模擬胃液環境中的存活性 52
第九節 噬菌體顆粒在模擬腸道中的釋放測試 55
結論 57
參考文獻 59
附錄 66
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