臺灣博碩士論文加值系統

黴漿菌在分類學上屬於Mollictes柔膜體綱,不具細胞壁被歸類為格蘭氏陽性菌，具有特別小的基因體，是目前在自然界中最小且可自行複製的原核生物。黴漿菌的細胞表面在與其宿主細胞的交互作用上扮演了重要的角色。豬肺炎黴漿菌(Mycoplasma hyopneumoniae)是豬黴漿菌性肺炎之病源菌，目前已知結合到豬氣管纖毛細胞是豬肺炎黴漿菌感染與繁殖的首要條件，並且會造成纖毛的脫落。本實驗以致病菌株strain 232與非致病菌株strain J來尋找可能之致病因子。我們以蛋白質二維電泳來比較 strain 232 與strain J之蛋白體差異，發現幾個在pI值與表現量具有明顯異的蛋白質P97、P110、P102 paralog與P216。其中P97是負責豬肺炎黴漿菌吸附到豬氣管纖毛細胞的蛋白質又稱黏蛋白(adhesin)，而P110與P216也是屬於類似功能的蛋白質(adhesin-like protein)。P102與P102 paralog與致病相關，在豬肺炎黴漿菌在感染豬氣管纖毛細胞時會有所表現。我們先將豬氣管纖毛細胞之蛋白質展開於二維電泳膠中，然後轉漬到硝化纖維紙上，接著與致病菌株strain 232之蛋白質反應。與豬氣管纖毛細胞蛋白質結合的黴漿菌蛋白。可以抗豬肺炎黴漿菌之抗血清(rabbit antiserum against Mycoplasma hyopneumoniae strain 232)偵測。發現豬氣管纖毛細胞蛋白lamin 和 peroxiredoxin，會與致病菌株strain 232之蛋白質結合。Lamin屬於一種絲狀蛋白，主要功能在於維持細胞核膜之骨架結構。Peroxiredoxin則牽涉到細胞中氧化還原反應的調節以及消除在代謝中產生的過氧化物。因此，我們推測peroxiredoxin可能藉由某種間接但尚未知的方式，參與了豬肺炎黴漿菌的致病機轉。

Mycoplasmas are a large group of diverse prokaryotic species comprising the class Mollicutes. Mycoplasmas lack cell walls, have remarkably small genomes, are phylogenetically related to gram-positive eubacteria, and are the smallest known self-replicating organisms. The surface of mycoplasmas clearly plays a critical role in the interaction of these organisms with their host cells. It is known that the initial event in colonization by Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia of swine, is binding to porcine tracheal ciliated cells and eventually caused the loss of cilia. The pathogenic strain 232 of Mycoplasma hyopneumoniae and an avirulent strain J were chosen to identify the possible virulence factors. We used 2D gel electrophoresis to compare the proteomic difference between stain 232 and strain J. The major differences are proteins P97, P110 and P216, each with varied pI and different degree of expression. P97 has been demonstrated as the adhesin molecule responsible for the adherence of Mycoplasma hyopneumoniae to the ciliated cells. Both P110 and P216 are the adhesin-like proteins. To investigate the interactions between Mycoplasma hyopneumoniae and porcine tracheal ciliated cells, we displayed the total protein of porcine tracheal ciliated cells in the 2D gel, transferred to NC paper and subsequently reacted with the total protein of Mycoplasma hyopneumoniae stain 232. The bound mycoplasmal proteins were then detected by the rabbit antiserum against Mycoplasma hyopneumoniae strain 232. Several tracheal ciliated cell proteins including lamin and peroxiredoxin, were found to react with the mycoplasmal proteins. Lamin belongs to the family of intermediate filaments, may be able to form a supportive nucleoskeletal structure underlying the nuclear envelope. Peroxiredoxin involves in redox regulation of the cell and in eliminating peroxides generated during metabolism. Therefore, it is anticipated that peroxiredoxin may play a role through a yet indirect and unknown pathway in the pathogenesis of porcine tracheal ciliated cells by the infection of Mycoplasma hyopneumonia.

中文摘要………………………………………………………………………………1
Abstract………………………………………………………………………………..2
壹、序論 …………………………………………………………………………….3
一、黴漿菌(Mycoplasma) ………………………………………………………..3
二、豬肺炎黴漿菌(Mycoplasma hyopneumoniae) ……………………………..5
三、實驗動機 ……………………………………………………………………8
貳、材料與方法 ……………………………………………………………………9
一、菌體培養 ……………………………………………………………………9
二、豬氣管纖毛細胞採集 ……………………………………………………..9
三、蛋白體分析－二維電泳 ……………………..……………………………10
1. 蛋白質樣品製備 ……………………….……………………………..10
2. TCA precipitation ………………………………………………………10
3. 蛋白質濃度測定 ……………………………………………………...11
4. 第一維等電點電泳(IEF) ……………………………………………...12
5. 二維電泳之SDS-PAGE展開 ..………………………………………..13
6. 銀染Silver Staining ...………………………………………………….14
7.二維電泳膠之影像分析 ..……………………………………………...15
四、M.hyopneumoniae 232與宿主細胞之作用 ………………………………16
五、In gel digestion(MS前處理)………………………………………………...18
六、MALDI-TOF 質譜分析……………………………………………………20
七、生物資訊分析………………………………………………………………21
叁、結果與討論 ……………………………………………………….22
一、比較Mycoplasma hyopneumoniae strain 232與strain J 之蛋白體 ………22
二、探討Mycoplasma hyopneumoniae strain 232與豬氣管纖毛細胞蛋白質之相互作用…………………………………………………………………………..25
肆、結論…………………………………………………………………27
伍、參考文獻……………………………………………………………29
附錄 ………………………………………………………………………………...60
附錄一、2 × Friis Medium製備(for M. hyopneumoniae strain 232) ………...60

附錄二、1 × Friis Medium製備(for M. hyopneumoniae strain J) …………...61
附錄三、質譜分析比對結果 ………………………………………………....62
附錄四、NCBI Blast比對結果 ……………………………………………….75
附錄五、CLUSTALW Multiple Sequence Alignment結果 …………………78
附錄六、TMHMM分析結果 ………………………………………………...85
附錄七、SignalP分析結果(100 a.a.) ..………………………………………...97









圖目錄
圖一、Mycoplasma hyopneumoniae strain 232與strain J之銀染二維電泳圖 …….34
圖二、Mycoplasma hyopneumoniae strain 232與strain J之coomassie blue染色二維電泳圖 ……………………………………………………………………………35
圖三、Mycoplasma hyopneumoniae strain 232與strain J 2DE gel A區域之平面及立體圖 ………………………………………………………………………………....36
圖四、Mycoplasma hyopneumoniae strain 232與strain J 2DE gel B區域之平面及立體圖 …………………………………………………………………………………37
圖五、Mycoplasma hyopneumoniae strain 232與strain J 2DE gel C區域之平面及立體圖 …………………………………………………………………………………38
圖六、Mycoplasma hyopneumoniae strain 232與strain J 2DE gel D區域之平面及立體圖 …………………………………………………………………………………39
圖七、Mycoplasma hyopneumoniae strain 232與strain J 2DE gel之 P102 paralog之平面及立體圖 …………………………………………………………………..40
圖八、M. hyopneumoniae strain 232之質譜分析二維電泳圖 ………………...…...41
圖九、M. hyopneumoniae strain 232之質譜分析二維電泳圖(圖二之2ED gel)…....42
圖十、M. hyopneumoniae strain J之質譜分析二維電泳圖(圖二之2ED gel)…….43
圖十一、M. hyopneumoniae strain 232之質譜分析二維電泳圖(小分子量)……….44
圖十二、豬氣管纖毛細胞蛋白質之銀染二維電泳圖 …………………………….45
圖十三、豬氣管纖毛細胞之質譜分析二維電泳圖 ………………………………..46
圖十四、Rabbit antiserum against Mycoplasma hyopneumoniae strain 232蛋白之靈敏度測試 ……………………………………………………………………………47
圖十五、Mycoplasma hyopneumoniae strain 232蛋白質與豬氣管纖毛細胞蛋白質結合測試(一維) ……………………………………………………………………..48
圖十六、Mycoplasma hyopneumoniae strain 232蛋白質與豬氣管纖毛細胞蛋白質結合測試(二維) ……………………………………………………………………..49
表目錄
表1、M. hyopneumoniae strain 232與strain J 2DE gel在A區域蛋白質點影像分析結果 …………………………………………………………………………………50
表2、M. hyopneumoniae strain 232與strain J 2DE gel在B區域蛋白質點影像分析結果 …………………………………………………………………………………51
表3、M. hyopneumoniae strain 232與strain J 2DE gel在C區域蛋白質點影像分析結果 …………………………………………………………………………………52
表4、M. hyopneumoniae strain 232與strain J 2DE gel在D區域蛋白質點影像分析結果 …………………………………………………………………………………53
表5、M. hyopneumoniae strain 232與strain J 2DE gel在E區域蛋白質點影像分析結果………………………………………………………………………..…………54
表6、M. hyopneumoniae strain 232二維電泳圖(一)質譜分析之結果……………..55
表7、M. hyopneumoniae strain 232二維電泳圖(二) 質譜分析之結果…………..56
表8、M. hyopneumoniae strain J二維電泳圖(二) 質譜分析之結果….……………57
表9、M. hyopneumoniae strain 232二維電泳圖(低分子量)質譜分析之結果...........58
表10、豬氣管纖毛細胞蛋白質譜分析二維電泳圖之結果……………………….59

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