臺灣博碩士論文加值系統

我們以本實驗室先前篩選到的具超廣效乙內醯胺酶（extended-spectrum β-lactamases, ESBLs）之克雷伯氏肺炎桿菌 (Klebsiella pneumoniae) 的一些菌株如K6、K55等， 和K. pneumoniae 10693作為噬菌體宿主細胞， 由醫院所收集的液體之上清液分離到12隻暴裂型噬菌體（KPP2，KPP3，KPP4，KPP5，KPP6，KPP7，KPP10，KPP11，KPP30，KPP42，KPP50和KPP95）， 並對其特性進行研究。 這些噬菌體的基本性質如titer（約109 PFU /ml）、限制酶 EcoRV 切取圖譜、核酸大小（約170-180 kb）均相似， 部分噬菌體外形結構也相似，推論他們屬同一類噬菌體。於是我們隨機取噬菌體KPP95 作進一步的特性研究。 KPP95 的titer約3 × 1010 PFU/ml， 能快速強烈的造成其宿主細胞K. pneumoniae 10693的裂解， 電顯外形上具有略長之二十面體的頭部， 接著連接含有一圈圈蛋白質外鞘， 其下再接一個基板。 KPP95的核酸大小約170-180 kb， 於G或C鹼基上被修飾並可能被醣化， 具低G+C組成。 這些性質均與T系列噬菌體相似。 有趣的是， 他不但可以感染許多具超廣效乙內醯胺酶的 K. pneumoniae， 也可感染1株抗cefotaxime的Klebsiella oxytoca 和1株具超廣效乙內醯胺酶的Enterobacter agglomerans， 為一泛宿主的噬菌體。 由KPP95核酸的選殖得到的之各核酸片段序列比對的結果， 顯示了 KPP95 與 T-even 或 pseudo-T-even 噬菌體較相似。 在蛋白質電泳分析下， 可看到 KPP95 約有 25個蛋白片段， 包含一個主要的蛋白片段約 46 kDa，此蛋白經N-端定序和基因23的PCR產物序列比對後， 顯示與T-even 或 pseudo-T-even 噬菌體的基因23轉譯出來的主要外套蛋白 (gp23) 相近。 此外， 藉由KPP95與其他 T4-type 噬菌體基因 18、19和23的比對結果， 發現他們的結構基因具有相似的設計形式。 從KPP95與其他17個 T4-type 噬菌體其基因23所建構出的種系發生樹狀圖， 我們推論 KPP95 應屬於 pseudo-T-even 噬菌體，而且與42噬菌體較為接近。

The KPP2, KPP3, KPP4, KPP5, KPP6, KPP7, KPP10, KPP11, KPP30, KPP42, KPP50 and KPP95 are twelve lytic bacteriophages. They were isolated from supernatant of hospital wastewater. These phages could lyse some of ESBLs (extended-spectrum β-lactamases) strains of Klebsiella pneumoniae (e.g. K6 or K55) and K. pneumoniae 10693. Under characteristic studies, they all were found to have similar basic properties such as titer (about 109 PFU/ml), restriction-cutting pattern of EcoRV, genome sizes (about 170-180 kb) and morphology of some these phages. We conclude that they are the same type of phages. KPP95 was randomized chosen for further characteristic studies and high titer of the phage (approximately 3 × 1010 PFU/ml) could be obtained. It could rapidly and violently lyse its host cell, K. pneumoniae 10693. Under electron microscope, it had morphology of a moderate elongated icosahedral head connected to a tail-sheathed tail and was terminated by a base plate. The genome size of KPP95 was estimated to be 170-180 kb. The DNA might have been modified on base G or C and have been glucosylated. The chemical composition of DNA of KPP95 possessed in a low G+C content situation. These properties are similar to T-type phages. Interestingly, KPP95 could also infect many ESBLs strains of K. pneumoniae, one cefotaxime-resisted Klebsiella oxytoca and one ESBLs strain of Enterobacter agglomerans. This suggests that it is a broad host range phage. The comparison of each KPP95 DNA fragments cloned from its genome also showed that KPP95 was much more similar to T-even or pseudo-T-even phages. SDS-polyacrylamide gel electrophoresis of the phage particles showed about 25 proteins. They included one major coat protein of 46 kDa. The N-terminal sequence of the major coat protein of KPP95 and PCR product sequence of its gene 23 were presented similarity to major coat protein (gp23) encoded by gene 23 of T-even or pseudo-T-even phages. Moreover, the alignments of gene 18, 19 and 23 of KPP95 revealed having similar modular or mosaic design with the structural genes of other T4-type phages. By the phylogenetic tree of the sequences of gene 23 of KPP95 and other 17 phages of T4-type, KPP95 should be belong to pseudo-T-even phages and much closer to phage 42.

目錄
中文摘要…………………………………………………………………………………1
英文摘要…………………………………………………………………………………2
前言………………………………………………………………………………………3
一、噬菌體的簡介…………………………………………………………………3
二、“protein antibiotics” 的發現………………………………………………… 6
三、噬菌體療法及其試驗…………………………………………………………7
四、克雷伯氏肺炎桿菌的簡介及其抗藥性………………………………………8
五、研究動機………………………………………………………………………9
材料與方法……………………………………………………………………………．11
I. 實驗材料…………………………………………………………………………．11
一、菌種、噬菌體與質體…………………………………………………………11
二、藥品與酵素……………………………………………………………………11
三、培養基…………………………………………………………………………11
四、試劑與緩衝溶液………………………………………………………………12
II. 實驗方法…………………………………………………………………………．14
一、細菌培養………………………………………………………………………14
二、菌種甘油保存法………………………………………………………………14
三、噬菌體的篩選…………………………………………………………………14
四、噬菌體濃度之測定……………………………………………………………14
五、噬菌體的純化…………………………………………………………………14
六、噬菌體之增殖…………………………………………………………………15
七、大量噬菌體液的製備…………………………………………………………15
八、K. pneumoniae 10693染色體DNA之抽取…………………………………15
九、噬菌體DNA之抽取…………………………………………………………16
十、照電子顯微鏡之前處理………………………………………………………16
十一、K. pneumoniae 10693生長曲線與細菌烈解曲線之測試…………………17
十二、噬菌體KPP95宿主分布的決定………………………………………… 17
十三、脈衝電泳法Pulsed-field gel electrophoresis（PFGE）………………… 18
十四、洋菜凝膠電泳製備與分析（agarose gel electrophoresis）………………18
十五、限制酶切割（Restriction enzyme digestion）……………………………19
十六、KPP95噬菌體其SDS-PAGE之蛋白分析……………………………… 19
十七、KPP95噬菌體蛋白質之N-端定序之處理……………………………… 20
十八、DNA的連接反應（ligation）…………………………………………… 21
十九、E. coli 之轉形作用（transfomation）……………………………………21
二十、小量質體DNA之抽取……………………………………………………21
二十一、聚合酶連鎖反應（Polymerase chain reaction）及其產物之定序…… 22
結果……………………………………………………………………………………．24
一、噬菌體的篩選…………………………………………………………………24
二、暴裂型噬菌體的基本性質……………………………………………………24
三、噬菌體KPP95之特性研究………………………………………………… 26
四、klebsiella pneumoniae 10693之染色體DNA的限制酶切割……………… 33
五、轉型株pOS 1、pOS 2、pOS 3其質體DNA的限制酶切割………………33
討論………………………………………………………………．……………………34
結論………………………………………………………………．……………………43
圖表………………………………………………………………．……………………45
參考文81
獻…………………………………………………………．……………………

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