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研究生:蘇資茜
研究生(外文):Zih-CianSu
論文名稱:探討困難梭狀桿菌潛力附著素Csp1之分子特性
論文名稱(外文):Molecular characterization of a putative adhesin Csp1 in Clostridium difficile
指導教授:黃一修黃一修引用關係
指導教授(外文):I-Hsiu Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:62
中文關鍵詞:困難梭狀桿菌附著素表面蛋白
外文關鍵詞:Clostridium difficileadhesinsurface proteins
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困難梭狀桿菌 (Clostridium difficile)是一種會產生內孢子的革蘭氏陽性、人類致病菌,通常與抗生素所造成的腹瀉相關。原本存在宿主腸道中的正常菌叢會因為施用廣效性抗生素而被破壞,這是導致困難梭狀桿菌感染 (CDI)的一個主要原因。在北美與許多的歐洲國家,高致病性菌株的出現以及高致死率,是導致CDI愈來愈嚴重。造成CDI相關症狀的主因為TcdA和TcdB兩種毒素,而在某些菌株中會表現二元體毒素CDT;另外,困難梭狀桿菌會表現非常多種表面蛋白,如S層蛋白 (S-layer proteins)、纖維黏連蛋白 (fibronectin-binding proteins) 和細胞壁蛋白 (Cwps)。目前已知革蘭氏陽性菌的表面蛋白在感染中扮演重要的角色,而分選酶是一種表現在細胞膜上的轉肽酶,會促進細胞表面蛋白共價鍵結至細胞壁。為了找出分選酶依賴性的表面蛋白在困難梭狀桿菌致病機制中所扮演的角色,透過生物資訊學分析找出困難梭狀桿菌630菌株中至少7種具有潛力的分選酶受質。在本研究中,我針對其中由CD2831所編碼的一種長度為972個胺基酸、有膠原蛋白結合功能的潛力細胞表面蛋白Csp1作為主要研究對象,此蛋白的PPKTG多胜肽顯示其可能為困難梭狀桿菌分選酶B的潛在受質。本研究的目的為探討Csp1的分子特性,建構了csp1插入性突變株KYC01,藉由Csp1多株抗體以免疫墨點分析法來確定Csp1的表現位置,但由於Csp1的低表現量,我在野生株與突變株大量表達Csp1,之後透過細胞分層實驗與免疫墨點法分析證明Csp1可以錨定在細胞壁上,有趣的是Csp1表達位置,主要在培養液中。以體外結合試驗,證明Csp1重組蛋白具有與膠原蛋白第一型結合的能力。由以上結果可以推論Csp1為困難梭狀桿菌潛力黏著素。
Clostridium difficile is a Gram-positive, endospore forming human pathogen and the most common cause of antibiotic-associated diarrhea within hospital settings worldwide. Disruption of the host’s indigenous microflora by broad-spectrum antibiotics is one of the major triggers for C. difficile infections (CDI). The emergence of hypervirulent C. difficile strains with high mortality rates have resulted in major epidemics in many parts of the world. C. difficile can express two exotoxins TcdA and TcdB, and in some strains a binary toxin CDT. Both TcdA and TcdB are potent toxins and are responsible for the extensive gastrointestinal inflammation and epithelial tissue damages found in an infected host. In addition, C. difficile is known to express a wide-variety of surface proteins such as S-layer proteins, fibronectin and cell wall proteins (Cwps). Surface proteins of Gram-positive bacteria are known to play important roles during infections. Sortase, a membrane anchored transpeptidase found ubiquitously in Gram-positive bacteria, promotes the covalent anchoring of surface proteins to the cell wall envelope. To determine the role of sortase-dependent surface proteins in C. difficile pathogenesis, I identified at least seven putative sortase substrates in C. difficile strain 630 by bioinformatics analysis. In this study, I focused on one of the putative cell surface proteins, Csp1. Csp1, encoded by CD2831, is a predicted protein of 972 amino acids and is annotated as a collagen-binding protein. The protein has a PPKTG motif suggesting that Csp1 could be a putative substrate of C. difficile sortase B. The aim of this study is to characterize molecular properties of Csp1. I constructed csp1 insertion mutant, KYC01, and defined the localization of Csp1 by immunoblotting using polyclonal anti-Csp1. Due to the low expression level of Csp1, I also overexpressed Csp1 in both CD630 and KYC01. Cellular fractionation and immunoblotting analysis demonstrated that Csp1 can be found anchored to the cell wall. Interestingly, the majority of Csp1 appear to be secreted into the medium. In-vitro binding experiments demonstrated the ability of Csp1 recombinant protein to adhere to Collagen I. In summary, the results generated from this project revealed a novel adhesin in C. difficile.
中文摘要 i
Abstract ii
誌謝 iv
目錄 vi
表目錄 x
圖目錄 xi
符號及縮寫 xii
1. 緒論 1
1.1. 困難梭狀桿菌簡介 1
1.1.1. 困難梭狀桿菌疾病 1
1.1.2. 流行病學 (epidemiology) 2
1.1.3. 困難梭狀桿菌菌株630 (Clostridium difficile 630) 2
1.1.4. 困難梭狀桿菌主要毒力因子 (Virulence factors) 2
1.1.5. 困難梭狀桿菌定殖 (colonization) 3
1.2. 分選酶簡介 4
1.2.1. 分選酶功能 4
1.2.2. 不同種類的分選酶 5
1.3. 生物資訊預測Clostridium difficile 630 潛力分選酶與潛力的分選酶基質 5
1.3.1. 困難梭狀桿菌潛力分選酶 (Putative sortase of Clostridium difficile) 6
1.3.2. 困難梭狀桿菌潛力的分選酶基質 (Putative sortase substrates) 6
1.3.2.1. CD0386與CD3392- 潛力結合膠原蛋白之表面蛋白 (putative collagen-binding surface proteins) 6
1.3.2.2. CD0183-潛力胞壁水解酶 (putative cell wall hydrolase) 6
1.3.2.3. CD3246-潛力表面蛋白 (putative surface protein) 7
1.3.2.4. CD2831-潛力膠原蛋白結合蛋白 (putative collagen-binding protein) 7
1.3.2.5. CD2537-潛力核苷酸酶與磷酸酯酶 (putative membrane-assocoiated 5’-ncleotidase/phosphoesterase) 7
1.3.2.6. CD3145-潛力絲胺酸-天門冬胺酸表面錨定纖維蛋白原結合蛋白 (putative serine-aspartate-rich surface anchored fribronogen-binding protein) 7
1.4. 研究動機 8
1.5. 研究目的 8
2. 材料與方法 9
2.1. 實驗菌株與動物來源 9
2.2. 質體來源 9
2.3. 實驗菌種培養與保存 9
2.4. DNA實驗操作 9
2.4.1. 商業化套件萃取質體DNA 9
2.4.2. 困難梭狀桿菌基因組DNA萃取 9
2.4.3. 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 10
2.4.4. 限制酶酵素切割 10
2.4.5. DNA黏合作用 (DNA Ligation) 11
2.4.6. 勝任細胞的製備 11
2.4.7. 大腸桿菌細胞轉型作用 11
2.4.8. 困難梭狀桿菌細胞轉型作用 12
2.5. RNA相關實驗 12
2.5.1. 萃取困難梭狀桿菌RNA 12
2.5.2. 反轉錄PCR (Reverse transcription-PCR, RT-PCR) 14
2.5.3. 即時定量聚合酶連鎖反應 (Real-Time quantitative PCR, Q-PCR) 14
2.6. 蛋白質相關實驗 14
2.6.1. 重組蛋白之表現與純化 14
2.6.1.1. 誘導重組蛋白大量表達 14
2.6.1.2. 親和性鎳離子螯合樹脂色層分析法純化蛋白 15
2.6.2. 蛋白質膠體電泳 15
2.6.3. 重組蛋白質身份鑑定樣品製備 16
2.6.4. 小鼠抗Csp1多株抗體血清製備 16
2.6.5. 困難梭狀桿菌蛋白質萃取 17
2.6.5.1. 困難梭狀桿菌細胞分層 (Cell Fractionation) 17
2.6.5.2. 蛋白質萃取 18
2.6.6. 免疫墨點法 (Immunoblotting) 18
2.6.7. 圓點墨點分析法 (Dot Blotting) 19
2.6.7.1. 樣品製備 19
2.6.7.2. 訊號偵測 19
2.6.8. 酵素連結免疫吸附分析法 (enzyme linked immunosorbent assay) 19
2.6.8.1. 偵測膠原蛋白第一型 (type I collagen) 19
2.6.8.2. 偵測Csp1重組蛋白 20
3. 結果 21
3.1. 構築csp1突變株 21
3.2. 構築csp1基因之互補株 22
3.3. 表現與純化Csp1重組蛋白 22
3.4. Csp1重組蛋白身份鑑定 (Protein identification) 23
3.5. 利用抗Csp1多株抗體比較野生株、突變株與互補株Csp1之表現 25
3.6. Csp1在困難梭狀桿菌中表現位置 26
3.7. Csp1重組蛋白與膠原蛋白第一型結合能力 27
4. 討論 28
5. 表 33
Table 1. Bacterial strains used in this study 33
Table 2. Plasmids used in this study 34
Table 3. Target sites identified by computer algorithm 35
Table 4. The primers used in this study 36
Table 5. Csp1 motifs identified by bioinformatic analysis 37
6. 圖 38
Fig. 1 Schematic representation of Csp1 (CD2831) protein organization with potential function domains. 38
Fig. 2 Schematic representation of ClosTron mutagenesis plasmid, pClostron2831. 39
Fig. 3 To check the PCR products of csp1 mutant by agarose gel electrophoresis. 40
Fig. 4 Plasmid map of pSS1. 41
Fig. 5 The analysis of Csp1 protein identification and purification. 43
Fig 6. The detection of Csp1 recombinant protein. 44
Fig. 7 The detection of Csp1 expression by immunoblotting in different strains. 45
Fig. 8 The cDNA of csp1 and CD2718 were detected by RT-PCR and Q-PCR. 46
Fig. 9 The phenotype of Csp1 expression. 47
Fig. 10 In-vitro binding experiments by ELISA 48
7. 參考文獻 49
8. 附錄 55
附錄一 55
附錄二 57
附錄三 58
附錄四 59




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