臺灣博碩士論文加值系統

尿素酶的活性在一些細菌中被視為毒性因子之一。在一些細菌中，尿素酶能夠分解尿素產生氨，使細菌能夠對抗環境中的酸性，例如幫助細菌在通過胃部時抵抗胃酸，或許是和腸內菌能達到腸道中定殖有關。一般來說，克雷伯氏肺炎菌（Klebsiella pneumoniae）在尿素酶活性測試中呈現陽性。我們分析本實驗室定序完成之克雷伯氏肺炎菌1084與克雷伯氏肺炎菌1158，在其中發現ure gene cluster 1以及ure gene cluster 2兩套疑似尿素酶基因組。在NCBI資料庫中已完成全基因體定序的64個克雷伯氏肺炎菌基因體中，不同血清型的菌株全部帶有 ure gene cluster 1。然而含有ure gene cluster 2的菌株僅佔了10.6%。在台灣血清型K1以及K2的克雷伯氏肺炎菌經常引起嚴重的感染，有鑑於此，我們特別針對由中山醫學大學得到的83株K1以及K2血清型之克雷伯氏肺炎菌臨床分離株進行ure gene cluster的PCR偵測與尿素酶活性實驗。結果顯示，在這些臨床分離株中，並不是所有的菌株都含有ure gene cluster 1。我們使用含有含尿素之培養基，進行尿素酶活性測試，臨床分離株中，大部分呈現紅色陽性反應。然而其中1158與1369都具備了兩套疑似尿素酶基因組，但是卻沒有尿素酶活性。相對的，其中有23個臨床分離株在PCR實驗中並沒有偵測到ure genes cluster的存在，卻依然在活性測試中呈現陽性。這個結果顯示這兩套疑似尿素酶基因組真正的功能很可能與尿素酶測試活性的結果無關。將來若能成功在克雷伯氏肺炎菌中構築這些疑似尿素酶基因組的剔除株，將能更加有助於我們釐清這些基因與尿素酶測試活性的關係，以及他們在克雷伯氏肺炎菌致病性中所可能扮演的角色。

The activity of urease is considered to be one of the virulence factors in some bacteria. Urease produced by bacteria decompose urea to produce ammonia, which neutralize the gastric acid produced by the host. As a consequence, this may enhance its intestinal colonization. Klebsiella pneumoniae is generally considered positive in urease activity test. We analyzed the genomic sequences of Klebsiella pneumoniae 1084 and Klebsiella pneumoniae 1158, two K1 and K2 clinical isolates previously sequenced by our group, and found two different putative urease gene clusters likely responsible for urease production, named ure gene cluster 1 and ure gene cluster 2. The ure gene cluster 1 can be found in all of the 64 K. pneumoniae complete genomes available in GenBank upto date. However, only 10.6% of them carried the ure gene cluster 2. K. pneumoniae K1 and K2 strains causes severe infections and were significant problem in Taiwan. To investigate the prevalence and the functions of these putative ure gene clusters among K1 and K2 strains, we carried out PCR detection and urease activity tests in 83 K. pneumoniae K1 and K2 isolates collected from Chung Shan Medical University Hospital. The results showed that all K1 strains were negeative for ure gene cluster 2. We performed a urease activity test using a culture medium containing urea and phenol red to detect the pH change. Almost all of the clinical isolatesare tested positive. Among these, 1158 and 1369 are PCR positive for both ure gene clusters. In contrast, 23 clinical isolates strain are positive in urease activity test, but PCR tested negative for both ure gene clusters. Our results suggest that the two putative urease gene clusters are likely not associated with the results of urease activity test. Further functional characterization will be necessary to clarify and confirm the roles of these genes in regarding to urease activity and bacterial pathogenesis.

中文摘要 i
Abstract ii
第一章 前言 1
第一節 克雷伯氏肺炎桿菌 1
第二節 克雷伯氏肺炎桿菌在臨床上造成的問題 2
第三節 克雷伯氏肺炎菌的致病因子 5
第四節 Urease的功能性基因體研究 6
第二章 研究目的 9
第三章 材料及方法 10
第一節 菌株、質體及生長環境 10
第二節 溶液配置 11
第三節 培養基等藥品配置 12
第四節 尿素酶活性測定 14
第五節 使用聚合酶鏈鎖反應（Polymerase Chain Reaction, PCR）檢測K.pneumoniae K1與K2血清型兩套Urease cluster 14
第六節 利用同源基因互換的方式建構克雷伯氏肺炎桿菌基因缺損的突變菌株 15
第七節 藍白篩選(blue and white screening) 18
第八節 質體分子選殖的快速篩檢 (Cracking) 18
第九節 抽細菌的質體DNA（QIAprep® Spin Miniprep kit） 19
第十節 利用限制酶（restriction enzyme）確認結果 19
第十一節 利用限制酶確認方向性 20
第十二節 將確認帶有ureA基因上下游片段的yT&A vector切限制酶並利用膠體電泳萃取純化 21
第十三節 DNA接合作用（Ligation） 22
第十四節 將yT&A vector上的ureA基因上下游的DNA片段轉移至自殺性質體pKAS46 23
第十五節 製作勝任細胞（competent cell） 25
第十六節 電穿孔（electroporation）實驗 25
第四章 結果與討論 27
一、 K. pneumoniae 1084 和K. pneumoniae 1158疑似尿素酶基因組（ure gene cluster）的註解與比較基因體學分析 27
二、 從已經有公開基因體序列的64個克雷伯氏肺炎菌的基因體資料分析疑似尿素酶基因組（ure gene cluster）的分佈，以及他們與各K型的關聯性 29
三、 以PCR偵測克雷伯氏肺炎菌臨床分離株中疑似尿素酶基因組（ure gene cluster）的分佈。 30
四、 克雷伯氏肺炎菌臨床分離株的尿素酶活性測試 32
五、 疑似尿素酶基因組之基因剔除株及基因補回株的建構，以及將來的功能性分析 34
第五章 未來的方向 36
參考文獻: 38
實驗圖表 43
圖1、尿素水解示意圖 43
圖2、K. pneumoniae 1084與1158之ure gene cluster 1 43
圖3、K. pneumoniae 1084與NTUH之ure gene cluster 1 44
圖4、K. pneumoniae 1084與KTCT2242之ure gene cluster 1 44
圖5、K. pneumoniae 1158與CG43之ure gene cluster 2 45
圖6、K. pneumoniae 1084與1158之ure 1/2 45
圖7、K1血清型之臨床分離株PCR偵測 46
圖8、K2血清型之臨床分離株PCR偵測 48
圖9、尿素酶活性測試12小時 50
圖10、尿素酶活性測試24小時 50
圖11、臨床分離株K1血清型尿素酶活性測試實驗 51
圖12、臨床分離株K2血清型尿素酶活性測試實驗 59
圖13、K1與K2血清型之三重覆實驗 65
圖14、同源性重組示意圖 73
圖15、針對ure gene cluster 1設計之引子 73
圖16、yt&A vector與P1以及P2方向關係 74
圖17、yt&A vector_P1與P2限制酶電泳分離圖 74
圖18、yt&A vector_P1P2 ligation示意圖 75
圖19、yt&A vector_P1以及yt&A vector_P2切限制酶電泳圖 76
表一、NCBI資料庫已完成定序之64株K. pneumoniae 77
表二、K1與K2血清型臨床分離株之PCR實驗結果 78
表三、臨床分離株尿素酶活性與PCR結果比較圖 79
附錄一、本論文設計之引子 80
附錄二、各菌種ure gene cluster比較 81
附錄三、尿素酶（Urease）受激活示意圖 82
附錄四、K-typing marker 83

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