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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 | Yung-Ching Fan | en |
dc.contributor.author | 范詠晴 | zh_TW |
dc.date.accessioned | 2021-06-16T02:48:16Z | - |
dc.date.available | 2020-09-25 | |
dc.date.copyright | 2015-09-25 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-07-16 | |
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Church, Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol, 1997. 179(20): p. 6228-37. 27. Tsai, C.M. and C.E. Frasch, A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem, 1982. 119(1): p. 115-9. 28. Blumenkrantz, N. and G. Asboe-Hansen, New method for quantitative determination of uronic acids. Anal Biochem, 1973. 54(2): p. 484-9. 29. Domenico, P., S. Schwartz, and B.A. Cunha, Reduction of capsular polysaccharide production in Klebsiella pneumoniae by sodium salicylate. Infect Immun, 1989. 57(12): p. 3778-82. 30. Hestrin, S., The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application. J Biol Chem, 1949. 180(1): p. 249-61. 31. Naran, R., G. Chen, and N.C. Carpita, Novel rhamnogalacturonan I and arabinoxylan polysaccharides of flax seed mucilage. 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Bera, A., et al., The presence of peptidoglycan O-acetyltransferase in various staphylococcal species correlates with lysozyme resistance and pathogenicity. Infect Immun, 2006. 74(8): p. 4598-604. 37. Ho, J.Y., et al., Functions of some capsular polysaccharide biosynthetic genes in Klebsiella pneumoniae NTUH K-2044. PLoS One, 2011. 6(7): p. e21664. 38. Islam, S.T. and J.S. Lam, Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway. Can J Microbiol, 2014. 60(11): p. 697-716. 39. Guo, H., et al., Molecular analysis of the O-antigen gene cluster of Escherichia coli O86:B7 and characterization of the chain length determinant gene (wzz). Appl Environ Microbiol, 2005. 71(12): p. 7995-8001. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54280 | - |
dc.description.abstract | 克雷伯氏肺炎桿菌為造成院內與社區感染的常見致病菌,莢膜是重要的致病因子,感染的病症及嚴重性和其莢膜型有相當的關聯。先前發現ref. K57和臨床菌株A1142以wzy基因分型法可分類為同莢膜型,但對其所分離的噬菌體卻有不同的辨識情形,發現在莢膜合成區中只有ref. K57有完整的轉醯酶(acetyltransferase)基因,後續證實在莢膜多醣上有乙醯基化修飾的差異,而確認莢膜型K57的亞型。 根據克雷伯氏肺炎桿菌K57莢膜亞型之發現與分析,wzy基因分型法歸類為K3及K54的臨床菌株1125-3及A35,其所分離的噬菌體對於標準菌株及臨床菌株有不同的辨識情形,因此想要進一步探討其莢膜合成區基因序列及莢膜結構之差異。首先將完整的莢膜型K54之標準與臨床菌株的莢膜合成區基因做比較,發現臨床菌株A35在轉醯酶出現提早的終止密碼子,基因大小較標準菌株K54縮短168個核苷酸。使用化學法及1H-NMR圖譜檢視ref. K54及A35之莢膜多醣乙醯基化是否有差異,發現兩者並無顯著差別。推測其早期終止密碼子並無影響其基因之活性區域,因此沒有發現莢膜多醣乙醯基之修飾差異,而後發現分離的ref. K54噬菌體為辨識脂多醣體,故噬菌體辨認不同。 另外,分析比對ref. K3及臨床菌株1125-3的莢膜合成基因,發現臨床菌株1125-3在莢膜聚合酶基因wzy出現提早的終止密碼子,使其wzy分為兩段;而標準K3菌株具有完整的wzy基因。因此建立1125-3 wzy置換菌株和1125-3 wzy基因剔除菌株,發現此兩株對噬菌體的辨識情形和1125-3野生株相同,並無改變;且莢膜表現量也無明顯差異。相對地,1125-3 wbaP基因剔除菌株的確沒有莢膜合成。wzy基因在莢膜合成上被認為扮演必要的角色,但使用南方墨點法並無發現在莢膜合成區外有與莢膜型K3 wzy相似之基因可替代其功能。推測特定莢膜型莢膜合成不一定需要wzy基因或其莢膜合成區外有另外的基因替代其功能,因此莢膜合成可能有另一套機制。 | zh_TW |
dc.description.abstract | Klebsiella pneumoniae is a major cause of nosocomial and community acquired infections. Its capsule is a virulence factor and capsular type is related to infection diseases and severities. Previous studies have shown ref. K57 and clinical strain A1142 using wzy genotyping could be classified into the same capsular type K57. However, phage typing showed different recognitions toward ref. K57 and A1142. Then our lab verified only that ref. K57 has a robust acetyltransferase gene in cps(capsular polysaccharide synthesis)region which can encodes an enzyme for capsule O-acetyl-modification. Hence, our lab confirmed that A1142 belongs to a subtype of capsular type K57. According to the finding of K57 subtype, this study focused on capsular type K3 strains (ref. K3 and 1125-3) and K54 strains (ref. K54 and A35) could be classified into the same genotype, but have different recognitions by phage typing. Their cps sequence and capsular structure were further studied. First, we compared the cps sequence of ref. K54 and its clinical strain A35 and found A35 has an early stop codon in acetyltransferase causing a truncated gene 168bp shorter than ref. K54. Investigation of O-acetylation amount on capsule by Hestrin’s method and 1H-NMR of capsular type K54 showed no distinct differences between two strains. Inferring the early truncation doesn’t influence the activity of acetyltransferase. Later, we discovered the phage of ref. K54 recognize LPS, not CPS, therefore resulting the different phage recognition between ref. K54 and A35. Second, we compared the cps sequences of the ref. K3 and its clinical strain 1125-3 and found 1125-3 has an early stop codon in wzy causing it separates into two parts. We constructed 1125-3 wzy replacement and deletion mutants, their capsule expression and phage recognition were analyzed accordingly. Replacement and deletion of wzy in 1125-3 did not decrease the capsule expression and did not alter the phage recognition, whereas deletion of wbaP caused capsule loss. Previous studies revealed wzy plays a putative polymerase role in capsule synthesis. However, no sequences similar to K3 wzy outside the cps region of 1125-3 were detected by Southern blotting. Our results indicated that may exist another gene or mechanism to compensate for wzy function in some capsular types. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T02:48:16Z (GMT). No. of bitstreams: 1 ntu-104-R02445122-1.pdf: 1097271 bytes, checksum: cfc66c5624298e54de295292b4ce1093 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 口試委員審定書 i 致謝 ii 中文摘要 iii 英文摘要 v 第一章、緒論 1 1.1 克雷伯氏肺炎桿菌基本介紹 1 1.2 克雷伯氏肺炎桿菌分型法與問題 2 1.3 克雷伯氏肺炎桿菌莢膜合成機制-wzy依賴途徑 3 1.4 克雷伯氏肺炎桿菌K57莢膜亞型之發現與分析 4 1.5 實驗目的-分析莢膜合成區序列及其莢膜結構和表現的關係 5 第二章、材料與方法 7 2.1菌株與載體 7 2.2 聚合酶鏈鎖反應(polymerase chain reaction, PCR) 7 2.3 建構無標記突變(unmarked mutation)菌株 7 2.4 噬菌體塗點實驗(spot test) 8 2.5免疫轉漬法(immunoblot) 8 2.6 銀染實驗(silver stain) 11 2.7 萃取克雷伯氏肺炎桿菌外圍醣類 12 2.8 酚-硫酸法(phenol-sulfuric acid assay) 13 2.9 硫酸定量莢膜多醣(CPS)含量 13 2.10 胞外多醣O-acetylation含量偵測 14 2.11 萃取克雷伯氏肺炎桿菌基因DNA(genomic DNA) 14 2.12 南方轉漬探針的合成 15 2.13南方轉漬分析(Southern blot): 16 2.14阿爾新藍染色(Alcian blue stain) 18 第三章、實驗結果 19 3.1 莢膜型K3及K54噬菌體分型辨識情形 19 3.2 莢膜型K54的丹麥標準菌株與臨床菌株A35之莢膜合成區基因比較 19 3.3 利用免疫墨點法偵測莢膜型K54菌株莢膜的表現 20 3.4 建立莢膜型K54之克雷伯氏肺炎桿菌wecA基因剔除菌株 20 3.5 莢膜型K54 wecA基因剔除菌株的莢膜多醣乙醯化表現型分析 21 3.6莢膜型K54 wecA基因剔除菌株之莢膜多糖O-acetylation官能基分析 21 3.7莢膜型K54 wecA基因剔除菌株之噬菌體分型辨識情形 21 3.8 莢膜型K3的丹麥標準菌株與臨床菌株1125-3之莢膜合成區基因比較 22 3.9 以塗點實驗測試臨床菌株1125-3之wzy基因置換菌株及剔除菌株之莢膜多醣表現型 22 3.10 以銀染偵測ref. K3、1125-3及突變株之脂多醣表現量 23 3.11 以阿爾新藍染色偵測ref. K3、1125-3及突變株之莢膜表現 23 3.12 利用免疫墨點法偵測突變株莢膜的表現型 24 3.13以硫酸法測定外膜多醣表現量 24 3.14 兩組引子之聚合酶連鎖反應探討1125-3、1125-3 wzy基因剔除菌株莢膜合成區外與wzy相似之基因 24 3.15 以南方墨點法偵測1125-3 wzy基因剔除菌株之莢膜合成區外與wzy相似之基因 25 3.16利用阿爾新藍染色法確認缺少wzy基因之菌株其莢膜表現量 25 第四章、討論 26 參考文獻 53 附錄 57 圖A、無標記突變株(unmarked deletion)篩選示意圖 57 圖B、無標記突變株(replacement)篩選示意圖 58 | |
dc.language.iso | zh-TW | |
dc.title | 克雷伯氏肺炎桿菌K3及K54之莢膜基因序列及化學結構分析 | zh_TW |
dc.title | Analysis of gene sequences and chemical structure of capsule polysaccharides in Klebsiella pneumoniae type K3 and K54 | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蔡丰喬(Feng-Chiao Tsai),吳世雄(Shih-Hsiung Wu) | |
dc.subject.keyword | 克雷伯氏肺炎桿菌,社區型化膿性肝膿瘍,莢膜型K54,莢膜型K3,聚合?Wzy, | zh_TW |
dc.subject.keyword | Klebsiella pneumoniae,community-acquired pyogenic liver abscess (PLA),capsular serotype K54,capsular serotype K3,polymerase Wzy, | en |
dc.relation.page | 58 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-07-16 | |
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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