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標題: | 以表現基因庫選殖幽門螺旋桿菌環丙沙星相關抗藥機制 Cloning and Characterization of Ciprofloxacin- Resistant Gene in Helicobacter pylori |
作者: | Juan-Ting Huang 黃絹婷 |
指導教授: | 王錦堂 |
關鍵字: | 幽門螺旋桿菌,喹,諾酮,環丙沙星, Helicobacter pylori,quinolone,ciprofloxacin, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 幽門螺旋桿菌 (Helicobacter pylori) 為外型呈螺旋狀、嗜微好氧環境的革蘭氏陰性桿菌。全世界約有50% 的受感染人口,此菌的感染也被發現與胃部癌症有顯著的相關性,因此世界衛生組織於1994年將幽門螺旋桿菌列為第一類致癌物質。目前隨著第一、二線藥物的菌株耐受性案例增加,臨床治療多選擇以喹諾酮類 (quinolone) 合併其他藥物做為第三線治療,但陸續有報導指出在歐亞地區的喹諾酮類抗藥菌株比例高達10-33%,並有逐年攀升的趨勢。先前研究發現此類抗藥菌株主要會在喹諾酮藥物的目標位置旋轉酶 (gyrase) 次單元- GyrA上的胺基酸67至106之間的區域有胺基酸的改變,致使藥物與旋轉酶間的親和力下降,藉此提高菌株的抗藥性,此區被定義為喹諾酮抗藥決定區域 (quinolone resistance-determining region, QRDR)。然而初步分析近年來於臺大醫院所收集的臨床抗藥菌株,發現約45%的喹諾酮類藥物-環丙沙星 (ciprofloxacin) 抗藥菌株並不具有GyrA QRDR的突變,這意味著可能存在其他的機制來調控菌株的抗藥能力。本實驗首先檢測台大醫院抗藥性菌株的最小抑菌濃度 (minimum inhibitory concentration, MIC),然後選擇NTUH-CIP3與11,最小抑菌濃度分別為8 μg/ml與2 μg/ml的抗藥性菌株,再分別定序gyrA與gyrB並利用自然轉型 (natural transformation) 的方法排除旋轉酶突變,最後選定一株NTUH-CIP3做為本實驗的主要研究材料。之後將主要研究菌株NTUH-CIP3之基因體進行部分酵解 (partial digestion),構築表現基因庫,保守估計此基因庫可達90%的涵蓋比例。之後以環丙沙星篩選基因庫,得到3個抗藥菌落,並定序此3株載體上的內插片段,皆涵蓋一段oorA的序列,之後為了評估oorA在抗藥機制上的重要性,利用自然轉型的方法將三株篩選自NTUH-CIP3的質體送入藥物感受性菌株HP26695當中,結果並沒有明顯提升感受性菌株的藥物耐受程度。因受限於表現基因庫本身的篩選限制,之後利用二維凝膠電泳分析臨床抗藥菌株,比較環丙沙星刺激前後的蛋白質體差異,挑選蛋白質表現差異4倍以上的蛋白質,以質譜分析後比對資料庫的結果,環丙沙星刺激下可能影響NTUH-CIP3的呼吸作用與異戊二烯 (isoprenoid) 類化合物的合成路徑。 Helicobacter pylori is a spiral gram-negative, microaerophilic bacterium, which causes type B gastritis and is associated with peptic ulcer. Furthermore, some studies have reported the association between H. pylori infection and the development of gastric carcinoma and lymphoma. Currently, the failure rate of H. pylori eradication with first or second-line therapy accelerated worldwide mainly due to H. pylori resistance to antibiotics. For this reason, quinolone-based triple therapy is used to eradicate H. pylori infection. Several studies have shown that the mutation in region between amino acids 67 to 106 (also called quinolone resistance-determining region, QRDR) of GyrA which is one of the gyrase subunits resulted in the decreased affinity between antibiotic and gyrase and enhanced the drug resistance. Analysis of ciprofloxacin (CIPRO)-resistant strains collected from National Taiwan University Hospital showed that∼45% resistant strains did not have mutations in GyrA QRDR, implying that there could be other mechanisms involved in quinolone resistance. Two strains, NTUH-CIP3 and 11 which minimum inhibitory concentration of CIPRO were 8 μg/ml and 2 μg/ml respectively, without gyrase mutations confirmed by gyrA and gyrB sequencing and natural transformation of gyrA and gyrB to a CIPRO susceptible strain were adopted in this study. The expression library of NTUH-CIP3 strain was constructed and utilized for identification of CIPRO resistance genes. The coverage of this library was estimated to 90%. Three different clones were selected from the library by CIPRO selection. All three clones covered oorA, a 2-oxoglutarate:acceptor oxidoreductase. In order to confirm the role of oorA in resistance to CIPRO, we transformed the 3 selected plasmids into CIPRO susceptible strain HP26695. Nevertheless, there was no CIPRO-resistant transformed strain. Because of the limitation of expression library, we used 2D-PAGE to compare proteome differences between CIPRO treated and non-CIPRO treated NTUH-CIP3. Then, we selected 4 fold increasing and decreasing spots, analysed the protein ID by MALDI Q-TOF, and blasting to protein databases. The protein data implied that the stimulation of quinolone might affect NTUH-CIP3 isopenoid-synthesis pathway and TCA cycle. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10031 |
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