臺灣博碩士論文加值系統

困難梭狀桿菌之毒素 A (TcdA) 及毒素B (TcdB)，是困難梭狀桿菌疾病致病的主要因素，由tcdA 與 tcdB 負責表達，該疾病通常與抗生素所造成的院內腹瀉相關。先前的研究發現TcdB 是決定困難梭狀桿菌感染病的重要因子。TcdA 及 TcdB 分別由 2710 及2366 胺基酸所組成，均有四個功能區域，分別是 glucosyltransfera-
ses domain (GTD)、cysteine protease domain (CPD)、translocation domain (TD)、combined repetitive oligopeptides (CROPs)。CPD 在 TcdB 自體蛋白水解過程中具有決定性的作用，通過自體蛋白水解的方式將GTD 水解並釋放到胞質溶膠細胞質中，GTD抑制Rho GTPase 使其失去活性，而引起細胞骨架受到破壞，最終導致細胞死亡。先前的研究提出，TcdB蛋白質序列之變異可能是高毒力株困難梭狀桿菌致病嚴重程度之重要決定因素。本實驗針對十一個來自高毒力株及一般菌株的TcdB CPD之自體蛋白水解活性進行研究。根據本實驗的蛋白質序列比對結果顯示，ribotype (RT) 012 (630)、RT 001 (ATCC9689)、RT 002 (TNHP20)、 RT 014 (TNHP68) 和 RT087 (ATCC43255) 並無差異。然而，屬於高毒力株的 RT 017 (TNHP82)，RT 027 (R20291) 及 RT 078 lineage胺基酸序列均有突變之現象，與一般菌株的胺基酸序列相似性百分比為 90.56 %、97.24 %、96.85 %。不僅如此，在體外水解活性試驗顯示，RT 017 (TNHP82) 和 RT 078 lineage在相同條件下，其自體蛋白水解活性更是比一般菌株來得高。 其中 RT 017 (TNHP82) 的自體蛋白水解活性rate為 0.94 μg/min， RT 078 lineage的自體蛋白水解活性rate 為0.16 μg/min，而一般菌株的自體蛋白水解活性rate 僅 0.07 μg/min。值得注意的是，RT 027 (20291) 具有最高的自體蛋白水解活性，該結果顯示於重組蛋白質純化結果。綜上所述，TcdB CPD 胺基酸之突變可影響並導致高毒力株和一般菌株之間自體蛋白水解速率差異。

Clostridium difficile (C. difficile) is the leading definable cause of antibiotic-associated diarrheal disease in hospitals due to its toxin A (2710 amino acids) and toxin B (2366 amino acids), encoded by tcdA and tcdB, respectively. Both consist of multiple function domains including glucosyltransferases domain (GTD), cysteine protease domain (CPD), translocation domain (TD), combined repetitive oligopeptides (CROPs). Previous study showed that TcdB is the key component of C. difficile virulent. CPD plays a decisive role in mediating autoprocessing and results in the release of GTD into the cytosol. GTD inactivates Rho family GTPase and leads to cytoskeleton disruption and eventual cell death. It has been reported that the variation in TcdB sequence could be in the hypervirulence strains that results an important determining factor in C. difficile associated disease severity. In this study, the autoprocessing activity of the TcdB from hypervirulent strains including ribotype (RT) 017 (TNHP82), RT 078 (M120), RT 027 (R20291) and historical stains including RT 012 (630), RT 001 (ATCC9689), RT 002 (TNHP20), RT 014 (TNHP68), and RT 087 (ATCC43255) were evaluated. TcdB CPD sequence analysis found that historical stains RT 012 (630), RT 001 (ATCC9689), RT 002 (TNHP20), RT 014 (TNHP68), and RT 087 (ATCC43255) share identical residues. However, the sequence homology of hypervirulent strains RT 017 (TNHP82), RT 078 lineage, and RT 027 (R20291), showed significant differences compared to historical stain which were 90.56 %, 96.85 %, 97.24 %, respectively. Moreover, in vitro cleavage assay has demonstrated that RT 017 (TNHP82) and RT 078 lineage possessed higher autoprocessing efficiency than others. The autoprocessing activity rate of RT 017 (TNHP82) was 0.94 µg/min. Simultaneously, the autoprocessing activity rate of other strains were 0.16 µg/min for RT 078 lineage strains and 0.07 µg/min for historical stains. It was notable that RT 027 (20291) had the highest autoprocessing activity, the result consistent with that observed during protein purification. Our data indicated that sequence variations in TcdB CPD of historical and hypervirulent strains could affect autoprocessing activity.

誌謝 i
中文摘要 ii
Abstract iii
目錄 iv
表次 vi
圖次 vii
第一章 前言 1
第二章 文獻探討 2
第一節 困難梭狀桿菌 2
一、困難梭狀桿菌背景概述 2
二、困難梭狀桿菌致病機制 2
三、困難梭狀桿菌疾病診斷標準 2
第二節 分子分型 (Molecular typing) 3
第三節 困難梭狀桿菌流行病學 4
一、人類流行病學 4
二、動物流行病學 4
三、高毒力株的出現 5
第四節 困難梭狀桿菌致病因素 7
一、困難梭狀桿菌致病機制之宿主因素 7
二、困難梭狀桿菌致病機制之毒力因子 7
第五節 困難梭狀桿菌之毒素 7
一、毒素蛋白Toxin A和Toxin B 7
二、毒素蛋白 Binary toxin 9
三、毒素蛋白Toxin A、Toxin B和Binary toxin 之致病機制 9
四、TcdB 為 CDI 的主要毒素蛋白 10
第五節　實驗動機與目的 10
第三章　材料與方法 12
第一節 實驗設計 12
第二節 困難梭狀桿菌之菌株 12
一、試驗藥劑及商業套組 12
二、困難梭狀桿菌之簡介 12
三、困難梭狀桿菌之培養 13
第三節 Genomic DNA之萃取 13
一、 試驗藥劑及商業套組 13
二、 基因萃取之酒精沉澱法 (ethanol precipitation of DNA) 13
三、 Genomic DNA 之純度分析與定量 14
第四節 tcdB fragment 之基因選殖 14
一、試驗藥劑及商業套組 14
二、tcdB fragment 轉殖基因及製備 15
三、表現載體 (vector) 之製備 16
四、限制酶水解 (restriction enzyme digestion) 16
五、接合作用 (ligation) 16
六、轉形作用 (transformation) 16
七、重組質體之篩選 17
八、定序及重組質體之保存 17
第五節 重組蛋白之表現、純化及分析 17
一、試驗藥劑及商業套組 17
二、選殖含接有目標基因之轉形至勝任細胞E. coli BL21 (DE3) 18
三、重組蛋白之大量表現及誘導 18
四、重組蛋白之純化 19
五、重組蛋白之分析、定量及保存 20
第六節 in vitro cleavage測試 21
一、試驗藥劑及商業套組 21
二、IPTG 誘導小量表現之比較 22
三、CPD蛋白質功能之活性分析 23
第七節 結果分析 23
第四章　實驗結果 24
第一節 一般菌株及高毒力株之 TcdB CPD 序列比對結果 24
第二節 tcdB fragment 之基因選殖結果 24
第三節 重組蛋白之表現、純化結果 25
第四節 in vitro cleavage 測試結果 26
一、重組蛋白誘導測試之比較 26
二、CPD 蛋白質功能之體外水解分析 26
第五章　討論 29
第一節 tcdB fragment之基因選殖結果探討 29
第二節 胺基酸序列比對結果探討 29
第三節 重組蛋白之表現結果探討 30
第四節 體外水解分析結果探討 30
第六章　參考文獻 33
附錄一 58
附錄二 68

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