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標題: | 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 Sequencing of CPS Region and Isolation of a Specific Bacteriophage of Capsular Type K56 of Klebsiella pneumoniae |
作者: | Yen-Hua Chen 陳彥樺 |
指導教授: | 王錦堂(Jin-Town Wang) |
關鍵字: | 克雷伯,肺炎桿菌,噬菌體,基因型, Klebsiella pneumoniae,bacteriophage,genotyping system, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 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 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22545 |
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顯示於系所單位: | 微生物學科所 |
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