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研究生:於冠芳
研究生(外文):Kuan Fang Yu
論文名稱:克雷伯氏肺炎桿菌毒性菌株Kp5莢膜基因組啟動子之調控
論文名稱(外文):Promoter studies of the cps gene cluster in a virulent klebsiella pneumoniae strain
指導教授:謝絹珠劉世東賴信志賴信志引用關係
指導教授(外文):J. H. WuS. T. LiuH. C. Lai
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:63
中文關鍵詞:克雷伯氏肺炎桿菌糖尿病
相關次數:
  • 被引用被引用:1
  • 點閱點閱:258
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  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
摘要
Klebsiella pneumonia 是人體腸道的常在菌,但在免疫低下的病人常造成疾病。近年來,由於院內感染Klebsiella pneumonia所導致的肝膿瘍更是常見。有研究指出,在國內因感染Klebsiella pneumonia所導致肝膿瘍的病患中,約有75﹪的病患同時伴隨著type Π DM。實驗發現,由肝膿瘍病患所分離的Kp5菌株,其cps region上共有4個具有啟動子活性的片段。其中位於orf2和wzi這兩個 open reading frames(ORFS)之間的啟動子B,活性在受到葡萄糖誘導後會大幅上升,其後的基因wzi之mRNA表現量也有明顯的增加。研究啟動子B之後基因wzi所轉譯出來的蛋白是否具有調控下游啟動子G之作用,結果發現下游的啟動子G,在啟動子B及wzi基因共同存在下,其活性會上升。在葡萄糖誘導下,啟動子G的活性也比單獨測得時來得高一些,但其後基因gmd、wcaG的表現量未增加,我們推測可能與葡萄糖溶液誘導下,莢膜中大量表現fucose而產生feedback inhibition有關。另外我們也發現,將Kp5菌種培養在含有葡萄糖溶液的LB broth中,其生長速率比養在未含有葡萄糖的LB broth中來的快。因此我們推測,細菌利用代謝葡萄糖產生能量,促進其本身生長繁殖之速率,及病患本身免疫力低下,是造成此菌對糖尿病患有高感染率的主要原因。
Abstract

Klebsiella pneumoniae is a normal flora present in human intestinal tract, but will cause disease in immunocompromised patients. Primary pyogenic liver abscess (PLA) caused by Klebsiella pneumoniae is an emerging infectious disease especially in diabetic patients. The effect of glucose concentrations on the promoter of capsular polysaccharide (cps) gene region was investigated. The putative promoters of the Kp5, a virulent strain, were cloned into a plasmid containing -galactosidase reporter system. Seven putative promoter regions, designated A, B, C, D, E, F, G, were cloned and the -galactosidase activity assays suggested that the regions of galF to orf2(A), orf2 to wzi(B), wzi to wza(G), and rfbp to gnd(E)have promoter activities. The promoter B in the upstream region of wzi showed higher promoter activity than others, enhanced by glucose addition, and wzi mRNA expression was elevated at the same time. To study transcription regulation of gene wzi, we observed that the activity of promoter G was elevated under the influence of promoter B and wzi gene. The over all activity of promoter G was increased under high glucose condition, however the gmd and wcaG mRNA expression was decreased, suggesting that the decrease of gmd and wcaG mRNA expression might be due to the feedback inhibition of the increased fucose final product. We also observed that Kp5 increased its growth rate at high glucose condition. In summary, our results suggested that high glucose condition, such as in the case of diabetes enhanced bacterial growth and the immunocompromised state of the DM patients were the major cause of diabetic patients with high susceptibility to Klebsiella pneumoniae infection.
目 錄
指導教授推薦書
口試委員會審定書
授權書………………………………………....................................iii
致謝……………………………………………….............................iv中文摘要........................………………………….…......................v
英文摘要……………………………………..……...........................vi
目錄………………………………………………...........................viii
圖表目錄......................................................................................ix
縮寫表...........................................................................................x
第一章 緒論............................................................................................1
第二章 研究動機.............................…………................................9
第三章 材料方法……………………….........................................11
第四章 實驗結果..................................………………..................20
第五章 實驗討論...........................…...........................................26
第六章 圖表................................................................................32
參考文獻......................................................................................48


圖表目錄
圖一:KP NTUH-k204菌株之莢膜合成相關基因組…...................4
圖二:KP致病相關毒力因子………………………………............32圖三:-galactosidase活性測定確定可能的promoter位置............33
圖四:不等濃度之葡萄糖溶液誘導 -galactosidase活性的改變......35 圖五:未做real time PCR前之RT-PCR檢視gmd、wcaG及wzi基因表現.........................................................................................37
圖六:Real time PCR分析環境中0mg/dl和400mg/dl葡萄糖溶液對gmd、wcaG及wzi基因表現的差異…………………………………38
圖七:promoter B與wzi基因共同調控下的promoter G之-galactosidase活性的改變……...................................................40
圖八:Kp5於環境中外加0mg/dl和400mg/dl葡萄糖溶液之生長曲線與活菌數…………………………………………….....................42
圖九:pKM005的限制酶圖譜.......................................................44
表一:本實驗所使用之菌種及質體................................................45
表二:本實驗所使用之單股核酸引子.............................................47
文獻探討
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