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研究生:鍾唯任
研究生(外文):Wei-Jen Chung
論文名稱:醱酵黴漿菌之蛋白體分析
論文名稱(外文):The proteome analysis of Mycoplasma fermentans
指導教授:林照雄林照雄引用關係周成功
指導教授(外文):Chao-Hsiung LinChen-Kung Chou
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
畢業學年度:92
語文別:英文
論文頁數:73
中文關鍵詞:醱酵黴漿菌蛋白體生物資訊學
外文關鍵詞:Mycoplasma fermentansproteomebioinformatics
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黴漿菌是目前已知一種最小的非共生細菌,他們擁有較其他細菌小(580-2200kb)且AT比例較高(67-76%)的基因體。到目前為止,已經有超過150種的黴漿菌被發現,他們廣泛分佈於各種動植物之間。醱酵黴漿菌(Mycoplasma fermentans)是這個研究的主角,透過之前的研究,已經知道它和類風濕性關節炎和後天免疫不全症候群等等一些人類免疫疾病密切相關,但是對於醱酵黴漿菌整個致病過程的瞭解卻還有待釐清。
在陽明基因體訂序中心初步完成醱酵黴漿菌基因體定序計畫之後,我們希望以生物資訊學和蛋白體學兩種方式對醱酵黴漿菌進行分析,希望對這株細菌有更進一步的瞭解。首先,利用GLIMMER可以預測出1027個開放讀取框架(Open Reading Frame,ORF)存在於醱酵黴漿菌的基因體中。另外,利用蛋白質序列的相似性,每個開放讀取框架的功能與其他黴漿菌的保存性(conservation)也被預測和建立起來。結果發現,屬於轉錄、DNA複製和醣類代謝這三種功能的蛋白質,在五種黴漿菌之中保存性最高。另一方面,為了預測醱酵黴漿菌的蛋白質交互作用,所以將醱酵黴漿菌的所有開放讀取框架序列與酵母菌(Saccharomyces cerevisiae)和幽門螺旋桿菌(Helicobacter pylori)的蛋白質作序列相似度比對,再參考這兩種細菌已知的蛋白質交互作用,就可預測出醱酵黴漿菌的蛋白質交互作用。最後有13個蛋白質交互作用被預測出來,而這些預測的蛋白質交互作用都和轉錄有關。
接下來,我們利用蛋白體技術去鑑定醱酵黴漿菌內表現出來的蛋白質,整個蛋白體都利用二維高壓液相層析法(2D-HPLC)進行分析,可溶的蛋白質部分則由二維電泳(2D-PAGE)進行分離,最後再利用質譜儀(mass spectrometry)鑑定。而到目前為止,已經有166個蛋白質被鑑定出來。由於醱酵黴漿菌擁有強烈的致病性,其可能致病的蛋白質:膜蛋白(membrane protein)或脂蛋白(lipoprotein),也是研究的重點。利用TopPred、TMHMM、SignalP和訊息序列(signal peptide)的保守性,一共預測出在醱酵黴漿菌的基因體中可能含有445個表現膜蛋白的基因和30個表現脂蛋白的基因。接下來,為了驗證預測的正確性,我們利用生物素(biotin)和清潔劑Triton X-114分別萃取出膜蛋白和脂蛋白。目前利用這兩種方法分別已經鑑定出17個和23個蛋白質。這些蛋白體的分析,不僅可以提供DNA或蛋白質序列之外的其他資訊,讓我們對於醱酵黴漿菌這個生命的運作有更進一步的瞭解,希望進一步還可以藉由這樣的研究找到治療醱酵黴漿菌相關疾病的可能治療方式。
Mycoplasmas are the smallest free-living bacteria. They all have small genome (580-2200 kb) with high percentage of AT content (67 to 76%). Nowadays, more than 150 species of mycoplasma have been discovered, and they are widely distributed in animals and plants. Mycoplasma fermentans have been reported to be involved in various human diseases, such as rheumatoid arthritis and AIDS. However, the pathogenic mechanisms of M. fermentans remain poorly understood. In addition to ongoing genome analysis of M. fermentans by YMGC (Yang-Ming Genome Center), we intended to analyze M. fermentans by bioinformatics and proteomics approach. From the sequenced genome of M. fermentans so far, 1027 ORFs were predicted by GLIMMER. The relationship between protein function and evolutional conservation was established through comparison of the protein sequence similarity of various mycoplasmas. From our observation, translation, DNA replication and carbohydrate metabolic machinery are the most conserved protein categories among mycoplasmas. Secondly, putative protein-protein interactions of M. fermentans were also predicted using current protein-protein interaction database of S. cerevisiae and H. pylori. However, only 13 protein-protein interactions, which are related to translation, were found. In addition to this, we also identified expressed ORF of M. fermentans using proteomic method. Whole proteome of M. fermentans was also identified by 2D-HPLC and all visible M. fermentans proteins on 2-D gel were identified with mass spectrometry. To date, 166 proteins have been identified. Due to the strong pathogenicity of M. fermentans, all potential pathogenic proteins, membrane proteins and lipoproteins, were predicted by TopPred, TMHMM, SignalP and signal peptide sequence similarity. Hundreds of proteins have predicted as membrane proteins, while 31 proteins are putative lipoproteins. For verifying the predictions, membrane proteins and lipoproteins were identified using biotin labeling and detergent extraction, respectively. 23 biotinylated proteins and 21 Triton X-114 extracted proteins have been identified. These proteomic analyses of M. fermentans have not only shown to provide a perspective into the activities of this organism beyond the obtainable by sequence alone, but also provided possible directions of treatment of M. fermentans-infected diseases.
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