Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21436
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dc.contributor.advisorShu-Fen Wengen_US
dc.contributor.advisor翁淑芬zh_TW
dc.contributor.authorChang, Hsiao-Chuanen_US
dc.contributor.author張曉娟zh_TW
dc.date2004zh_TW
dc.date.accessioned2014-06-06T07:15:48Z-
dc.date.available2014-06-06T07:15:48Z-
dc.identifier.urihttp://hdl.handle.net/11455/21436-
dc.description.abstract我們從醫院廢液樣品中分離到 8 株可以感染 Stenotrophomonas maltophilia 的噬菌體,命名為 SMA1 ~ SMA8。 其中 SMA2、SMA5、SMA6 具有較強的溶菌能力。 將噬菌體 DNA 以限制酶切割後,電泳分析比對得知,SMA2,SMA5,SMA6 具有相同的限制酶切割圖譜,推測三者應為相同或極為相似之噬菌體。 因此針對 SMA5 進行特性分析。 在電子顯微鏡下觀察噬菌體外型,得知 SMA5 屬於噬菌體分類中的 Myoviridae。 由於多種限制酶皆無法切割噬菌體,推測噬菌體 DNA 可能存在不尋常之修飾。 利用脈衝式電泳法分析 SMA5 基因體,得知基因體大小約為 250 kb。 噬菌體結構蛋白及 N 端定序分析顯示 SMA5 為一株全新的大型噬菌體。 利用 spot test 測試 SMA5 之宿主範圍及 SMA5 對 S. maltophilia 的感染情形,由結果得知在受測的臨床分離株中有 94% S. maltophilia 分離株受 SMA5 感染後產生溶菌圈,此外 SMA5 亦可感染 2 株 E. coli 及 2 株 Serratia marcescens 的分離株。 測試噬菌體 SMA5 保存十六個月的噬菌體液之效價,由效價下降的幅度有限,顯示 SMA5 是一株穩定的噬菌體。 另一方面,在 S. maltophilia 分離株的上清液發現有噬菌體存在,推測這些噬菌體可能屬於 temperate phage,命名為 SMT 系列。 純化其中一株噬菌體 SMT13,利用限制酶切割片段次選殖方式,將 SMT13 基因體定序完成。 噬菌體 SMT13 基因體大小為 33,525 bp,經由電腦分析比對,推測有 43 個 ORFs 存在。 利用南方轉漬法結果推測 SMT13 之 DNA 以環形存在。 由蛋白分析比對,基因組成與排列順序,及外型等多項特徵顯示 SMT13 與 Pseudomonas aeruginosa phage CTX 及 phage P2 相似,推測同屬 P2-like phage。 此外,利用 NCBI-blastx 分析比對發現 SMT13 與 X. campestris pv. campestris ATCC 33913 之 prophage XccP1 及 X. axonopodis pv. citri 306 之 prophage XacP1 在溶裂週期所需之基因 (噬菌體結構相關基因與 lysis 基因) 相似性高,但與噬菌體 DNA 複製及潛溶週期相關基因差異性變大且有缺失及位移的現象,推測此部分基因的差異影響 prophage 的釋出能力。 除了 coliphage 186 外,大多數的 P2-like phage 皆無法被外來因子誘導釋出噬菌體。 但利用 mitomycin C 成功的誘導 S. maltohlilia T13 釋出噬菌體 SMT13,推測 S. maltophilia 噬菌體釋出調控機制與大多數 P2-like phage 相異,值得進一步深入研究。zh_TW
dc.description.abstractEight strains of lytic phages, including SMA1 ~ SMA8, were isolated from 87 strains of S. maltophilia that were isolated from wastewater and catheter-related devices. SMA2, SMA5, and SMA6 could lyse most clinical isolates of S. maltophilia. These phages contain a double-strained DNA genome with a similar restriction pattern. Electron microscopy shows that SMA5 belonges to the family of Myoviridae. Pulsed-field gel electrophoresis shows that SMA5 has a genome of about 250 kb. N-terminal sequencing of four virion proteins reveals that SMA5 is a novel phage. Various S. maltophilia isolates were used for infection with SMA5, and varied degrees of growth inhibition were observed. No significant loss of phage infectivity was observed after storage of crude lysates of SMA5 at 4℃ for 16 months, indicating that SMA5 is stable. In the other way, it was found that 46 out of 87 collected S. maltophilia strains were able to release infective phage particles. DNA sequencing reveals that the genome of SMT13 is circular, with 33,525 nt in size. Forty-three open reading frames (ORFs) were identified on the SMT13 genome. Based on comparative the analysis of sequence conservation, genome organization, and morphology it is believed that SMT13 is similar to P2 and P2-like phages. Electron microscopy of SMT13 confirmed that this phage belonges to Myoviridae. In order to illustrate the phylogenetic relatedness, SMT13, prophage XccP1, prophage XacP1, P2 and P2-like phages were analyzed with programs BLAST, ClustalX, TreeView, TMHMM, Dot plot, SignalP, and GCG. The part of SMT13 genome containing genes coding for structural proteins, assembly proteins, and terminase is highly related to the P2 and P2-like phages. The other part of the SMT13 genome containing genes coding for lysis related proteins, DNA replication related proteins, lysogeny regulators is far related to the P2 and P2-like phages. The phylogenetic relatedness between SMT13 and prophages on the gamma Proteobacteria genome, including Xylella, Xanthomonas, Stenotrophomonas, and Pseudomonas, was further analyzed.en_US
dc.description.tableofcontents縮寫字對照表............................ 1 中文摘要.............................. 2 英文摘要.............................. 3 壹、前言.............................. 4 貳、材料與方法...........................10 I. 實驗材料 ...........................10 II. 實驗方法 ...........................14 一、細菌之培養.........................14 二、噬菌體的篩選方法......................14 三、噬菌體的純化........................14 四、噬菌體的培養........................15 五、噬菌體顆粒濃度 (titer) 的測定 ................15 六、點測試驗 (spot test) .....................15 七、Plaque assay ........................16 八、Liquid assay ........................16 九、DNA 之製備 ........................16 十、洋菜膠體電泳分析......................18 十一、脈衝式電泳分析......................18 十二、質體之選殖........................19 十三、南方轉漬法........................20 十四、蛋白質凝膠電泳分析....................22 十五、噬菌體蛋白質N端定序之處理 ...............23 十六、生長曲線測定.......................23 十七、穿透式電子顯微鏡樣品之前處理...............24 參、結果與討論 第一部份:S. maltophilia 溶裂型噬菌體之分離及特性分析 .........25 一、S. maltophilia 菌株之基本生理特性 .................25 1. 最適生長溫度 .........................25 2. 外型 (morphology) .......................25 3. 菌數及生長世代 (generation time) ................25 二、S. maltophilia溶裂型噬菌體之分離與純化 ..............25 三、S. maltophilia溶裂型噬菌體 SMA5 之特性分析 ...........26 1. 限制酶切割圖譜及基因體大小 (genome size) ............26 2. 噬菌體外型 (morphology) ....................27 3. 病毒體結構蛋白質分析及蛋白 N 端定序 (N-terminal sequencing) ...28 4. 宿主分佈 (host range) .....................29 5. 噬菌體 SMA5 之穩定性 ....................29 6. S. maltophilia 之生長曲線與細菌裂解 (bacteriolysis) 曲線 ......30 7. 噬菌體 SMA5 之應用與展望 ..................31 第二部份:S. maltophilia潛溶性噬菌體之分離、基因體定序及特性分析 ...33 一、S. maltophilia潛溶性噬菌體之分離與純化 ..............33 二、S. maltophilia 潛溶性噬菌體 SMT13 之特性分析 ...........33 1. 噬菌體外型 ..........................33 2. 蛋白質分析及宿主分佈 (host range) ................34 3. 利用 chloroform 破菌後,對 SMT13 噬菌體釋出的影響,及以 mitomycin C (MMC) 誘導 SMT13 噬菌體之釋出 .........34 4. 潛溶性噬菌體 SMT13 基因體之定序與分析............36 三、S. maltophilia 潛溶性噬菌體 SMT13 之演化分析 ...........58 1. SMT13 與 P2 及 P2-like phage .................58 2. SMT13 與 Xanthomonas 基因體上的潛溶性噬菌體 ........60 肆、參考文獻.............................63 伍、圖表...............................71 陸、附錄............................. i — xxvzh_TW
dc.language.isoen_USzh_TW
dc.publisher分子生物學研究所zh_TW
dc.subjectphageen_US
dc.subject噬菌體zh_TW
dc.subjectStenotrophomonas maltophiliaen_US
dc.subject院內感染菌zh_TW
dc.titleIsolation and characterization of Stenotrophomonas maltophilia phages SMA5 and SMT13en_US
dc.titleStenotrophomonas maltophilia 噬菌體 SMA5 及 SMT13 之分離與分析zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
item.cerifentitytypePublications-
item.grantfulltextnone-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.languageiso639-1en_US-
Appears in Collections:分子生物學研究所
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