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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22545完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂(Jin-Town Wang) | |
| dc.contributor.author | Yen-Hua Chen | en |
| dc.contributor.author | 陳彥樺 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:20:26Z | - |
| dc.date.copyright | 2010-09-09 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-07-20 | |
| dc.identifier.citation | 1. Bryan CS, Reynolds KL, Brenner ER. Analysis of 1,186 episodes of
gram-negative bacteremia in non-university hospitals: the effects of antimicrobial therapy. Rev Infect Dis. 1983, 5(4):629–638. 2. R C Bone. Gram-negative sepsis: a dilemma of modern medicine. Clin Microbiol Rev. 1993, 6(1): 57-68 3. Abbott SL. Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and other Enterobacteriaceae. In: Murray PR, Baron EJ, Jorgensen JH,Pfaller MA, Yolken RH, eds. Manual of clinical microbiology. 8th ed.Washington DC: American Society for Microbiology Press, 2003:684–700. 4. Eisenstein BI, Zaleznik DF. Enterobacteriaceae. In: Mandell JEB, Dolin R, eds. Principles and practice of infectious diseases. 5th ed. Philadelphia:Churchill- Livingstone, 2000:2294–2309. 5.. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors.Clin Microbiol Rev 1998; 11:589–603. 6. Wacharotayankun R, Arakawa Y, Ohta M, et al. Enhancement of extracapsular polysaccharide synthesis in Klebsiella pneumoniae by RmpA2, which shows homology to NtrC and FixJ. Infect Immun 1993; 61: 3164–3174. 7. Mizuta K, Ohta M, Mori M, Hasegawa T, Nakashima I, Kato N. Virulence for mice of Klebsiella strains belonging to the O1 group: relationship to their capsular (K) types. Infect Immun 1983; 40:56–61. 8. Pan YJ, Fang HC, Yang HC, Lin TL, Hsieh PF, Tsai FC, Keynan Y, Wang JT.Capsular polysaccharide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype. J Clin Microbiol. 2008, 46(7):2231-2240. 9. Durlakowa I, Lachowicz Z , Slopek S. Serologic characterization of Klebsiella bacilli on the basis of properties of the capsular antigens. Arch Immunol Ther E.xp 1967; 15:(4):497-504. 10.Palfreyman JM. Klebsiella serotyping by counter-current immunoelectrophoresis. J Hyg 1978, 81:219-225. 11. P. Pieroni, R. P. Rennite, B. Ziola and H. G. Deneer. The use of bacteriophages to differentiate serologically cross - reactive isolates of Klebsiella pneumoniae. J. Med. Microbiol. 1994, 41: 423-429 12. Combe ML, Pons JL, Sesboue R, Martin JP. Electrophoretic transfer from polyacrylamide gel to nitrocellulose sheets, a new method to characterize multilocus enzyme genotypes of Klebsiella strains. Appl Environ Microbiol 1994; 60 :26-30. 13. Costas M, Holmes B, Sloss LL. Comparison of SDS-PAGE protein patterns with other typing methods for investigating the epidemiology of ' Klebsiella aerogenes'. Epidemiol Infect. 1990, 104: 455-465 14. Sylvain Brisse, Sylvie Issenhuth-Jeanjean, and Patrick A. D. Grimont. Molecular Serotyping of Klebsiella Species Isolates by Restriction of the Amplified Capsular Antigen Gene Cluster. J Clin Microbiol. 2004, 42(8):3388-3398. 15. Dietlinde Rieger-Hug and Stephan Stirm. Comparative Study of Host Capsule Depolymerases Associated with Klebsiella Bacteriophages. Virology 1981, 113: 363-378 16. Chi-Jen Lee and Kalo Koizumi. Immunochemical relations between pneumococcal group 19 and Klebsiella capsular polysaccharides.The Journal of Immunology 1981,127 (4):1619-1623 17. Guy G.S. Dutton et. al. Preparation of oligosaccharides by the action of bacteriophage-borne enzymes of klebsiella capsular polysaccharides. Carbohydrute Research, 1981,97:127-138 18. Guy G.S. Dutton, Haralambod Parolis. The use of bacteriophage depolymerization in the structural investigation of the capsular polysaccharide from Klebsiella serotype K3. Carbohydr Res. 1986,149(2):411-23. 19. Merrifield EH, Stephen AM. Structural investigation of Klebsiella K-type 4 capsular polysaccharide. Carbohydr Res., 1981 Oct 1;96(1):113-20. 20. Elsasser-Beile U, Friebolin H, Stirm S.Primary structure of Klebsiella serotype 6 capsular polysaccharide Carbohydrate Research. 1978,65:245-249 21. Kjellberg A,Widmalm G,Jansson P,Nimmich W.Structural studies of the capsular polysaccharide from Klebsiella type 7.Carbohydrate Research. 1995.273 (1): 53-62 22. Erbing B,Jansson P,Widmalm G,Nimmich W. Structure of the capsular polysaccharide from the Klebsiella K8 reference strain 1015. Carbohydrate Research. 1995,273 (2):197-205 23 Cescutti P, Paoletti S. On the specificity of a bacteriophage-borne endoglycanase for the native capsular polysaccharide produced by Klebsiella pneumoniae SK1 and its derived polymers. Biochemical and Biophysical Research Communications 1994,198:1128-1134 24. Mukherjee A, Roy N. Synthesis of methyl 3-O-[3-O-( | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22545 | - |
| dc.description.abstract | 克雷伯氏肺炎桿菌是造成院內與社區感染的常見致病菌，其莢膜已被報導為重要的致病因子之ㄧ。過去克雷伯氏肺炎桿菌分類以莢膜血清型為主，然而許多臨床報告已顯示，多種血清都與一種以上的莢膜多醣反應，影響鑑別的準確性。本實驗室已發展以聚合 | zh_TW |
| dc.description.abstract | Klebsiella pneumoniae is a major cause of nosocomial and community- acquired infections, and its capsule has reported to be a virulence factor. There were reports of cross reactivations that occur among the defined 77 capsular serotypes by using anti-sera. Our lab has developed a cps-PCR genotyping to deduce serotyping upon the cps sequences were available. Recently, we also explored the specificity and sensitivity for capsular typing by bacteriopahges. Therefore, resolving the cps sequences of all reported capsular types and isolation of capsular-type-specific bacteriophages will provide a accurate and easier method for capsular typing.
In this study, we completed sequencing of a cps synthesis region and isolated a bacteriophage of capsular type K56 of K. pneumoniae. This region composed of 16 ORFs, including 7 conserved CPS synthesis genes:galF, ORF2, wzi, wza, wzb, wzc, and gnd; 4 putative glycosyltransferases ;a putative glycosylhydrolase; and a putative pyruvyl transferase. Cps-PCR genotyping by primers designed on ORF12 was specific for capsular type K56. Then, a new bacteriophage was isolated from sewage, and exhibited a host range-restricted to the K. pneumoniae type K56 reference strain. . Full genome sequence reveled highly similarity, about 90% identity in amino acid level, to our previously isolated K1 phage. Sequence anlysis showned the CPS-depolymerase may form a fusion protein with the tail spike protein. Combining the methods of conserved and hypervariable sequence identification, we also developed a novel genotyping system based on sequencing hypervariable region of wzc after amplification by PCR using primers designed on conserved regions of wza and wzc. More than 80% of the reference strains can be amplified by one pairs of conserved primer; in addition, with 4 helper primers, about 95% strains amplified by 5 primer pairs. Except 2 strains (K15 and K50), the sequence of all reference strains have been built in our database and showed uniqueness. The capsular types of 32 non-tissue-invasive strains collected from 1997 to 2003 are determined by this method：24 strains have wzc homologues in reference strains, 7 strains which didn’t match any sequence pattern in the database, and one failed to be amplified by our defined primer pairs. The specificity of this method will be further evaluated. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:20:26Z (GMT). No. of bitstreams: 1 ntu-99-R97445102-1.pdf: 2069959 bytes, checksum: a3de12ebfc6af4ce16747404717dd78a (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 圖目錄.........................................................3
表目錄.........................................................4 中文摘要.......................................................5 英文摘要.......................................................6 第一章、緒論...................................................8 第二章、材料與方法............................................12 1.實驗菌株..............................................12 2.質體與培養基..........................................12 3. LA-PCR擴增克雷伯氏肺炎桿菌莢膜型K56莢膜合成區基因.12 4. 限制性核酸內切酶實驗.................................14 5. DNA選殖(DNA cloning) ................................14 6. 定序(sequencing)與序列連接(contigue) ....................15 7. 建構克雷伯氏肺炎桿菌莢膜型K56莢膜合成基因 醣基轉移酶突變株.....................................16 8. 異位互補實驗.........................................17 9. 熱酚水法抽取莢膜.....................................18 10.狹縫點墨法(slot blot) ..................................19 11.尋找及分離克雷伯氏肺炎桿菌莢膜型K56的噬菌體.........20 12.塗點實驗(spot test) .....................................21 13.噬菌體增殖(phage amplification) ..........................21 14.噬菌體純化與DNA萃取................................22 15.噬菌體DNA選殖(DNA cloning)與定序....................23 16. 十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE) 與西方轉漬(Western blot) ............................24 17.噬菌體降解莢膜多醣實驗............................. .25 18.莢膜合成基因區的生物資訊分析.........................26 19. 高通量定序(high throughput sequencing) ..................27 第三章、實驗結果 1. 克雷伯氏肺炎桿菌莢膜型K56莢膜合成基因區定序.........28 2. 無標記基因剔除法與異位互補實驗確認此基因區對 莢膜合成的影響......................................29 3. K56莢膜型聚合酶鏈反應引子對..........................29 4. 尋找及分離克雷伯氏肺炎桿菌莢膜型K56的噬菌體.........29 5. 噬菌體DNA萃取......................................30 6. 噬菌體基因定序.......................................30 7. 噬菌體宿主範圍與專一性實驗...........................31 8. K56噬菌體感染宿主能力與宿主莢膜存在有關.............31 9. K56噬菌體具有降解K56莢膜的能力....................31 10.克雷伯氏肺炎桿菌基因型鑑定系統之研發.................32 第四章、討論 1.克雷伯氏肺炎桿菌莢膜型K56莢膜多醣結構與合成基因區.....36 2. 克雷伯氏肺炎桿菌噬菌體親源分析........................36 3. 比較新分離的K56噬菌體與Φ56噬菌體...................38 4. 克雷伯氏肺炎桿菌基因型鑑定系統之研發..................39 5. 蛋白質保守序列分析....................................39 6. 引子位於功能性序列....................................40 7. 序列相似度與莢膜多醣結構之關聯........................41 第五章、總結...................................................42 第六章、參考文獻...............................................75 圖目錄 圖一、 77種莢膜血清型構造關係圖...............................44 圖二、 克雷伯氏肺炎桿菌莢膜型K56的莢膜合成區基因圖譜.........45 圖三、 限制性核酸內切酶實驗...................................46 圖四、 莢膜型K56 莢膜合成區ORF8-11無標誌基因剔除、異位互補..47 圖五、 各水源分離之噬菌體塗點實驗.............................48 圖六、 分離之K56噬菌體的限制片段長度多態性(RFLP)............49 圖七、 K56噬菌體基因體圖譜...................................50 圖八、 KP56噬菌體與KP1噬菌體基因體比較.....................51 圖九、 K56噬菌體宿主範圍測試.................................52 圖十、 K56噬菌體感染克雷伯氏肺炎桿菌與莢膜的關係.............53 圖十一、K56噬菌體降解K56莢膜實驗.............................54 圖十二、比較K1與 K56噬菌體溶菌斑大小.........................55 圖十三、克雷伯氏肺炎桿菌莢膜合成基因區重組區塊分析.............56 圖十四、突變比率分析(non-synonymous / synonymous) ...............57 圖十五、核酸多型性(Nucleotide polymorphism) .....................58 圖十六、wza與wzc的核酸多型性.................................59 圖十七、PCR確認wzc高度變異區於相同血清型中的保守性..........60 圖十八、Wza的蛋白質間作用界面與保守引子位置...................61 圖十九、Wzc酪胺酸激酶蛋白質結構域與保守引子位置...............62 圖二十、Wzc的蛋白質間作用表面與保守引子位置...................63 圖二十一、比較78種莢膜型代表菌株與非組織侵襲性臨床分離菌株 中保守引子使用率(primer pairs preference) ..................64 圖二十二、比較肝膿瘍菌株與非組織侵襲性臨床分離菌株分型.........65 圖二十二、無標記基因剔除法實驗流程.............................66 表目錄 表一、研究中使用的細菌菌株及質體...............................67 表二、研究中所使用的引子.......................................69 表三、克雷伯氏肺炎桿菌莢膜型K56莢膜合成區 開放閱讀框生物資訊分析...................................71 表四、克雷伯氏肺炎桿菌莢膜型K56噬菌體KP-K56基因體 開放閱讀框生物資訊分析...................................72 | |
| dc.language.iso | zh-TW | |
| dc.subject | 基因型 | zh_TW |
| dc.subject | 克雷伯 | zh_TW |
| dc.subject | 肺炎桿菌 | zh_TW |
| dc.subject | 噬菌體 | zh_TW |
| dc.subject | genotyping system | en |
| dc.subject | Klebsiella pneumoniae | en |
| dc.subject | bacteriophage | en |
| dc.title | 克雷伯氏肺炎桿菌莢膜型K56
莢膜合成基因定序與噬菌體分離 | zh_TW |
| dc.title | Sequencing of CPS Region and Isolation of a Specific Bacteriophage of Capsular Type K56 of Klebsiella pneumoniae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 賴信志(Hsin-Chih Lai),鄧述諄(Shu-Chun Teng) | |
| dc.subject.keyword | 克雷伯,肺炎桿菌,噬菌體,基因型, | zh_TW |
| dc.subject.keyword | Klebsiella pneumoniae,bacteriophage,genotyping system, | en |
| dc.relation.page | 89 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-07-20 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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