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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 王錦堂(Jin-Town Wang) | |
dc.contributor.author | Ching-Ching Chen | en |
dc.contributor.author | 陳青青 | zh_TW |
dc.date.accessioned | 2021-06-16T10:20:30Z | - |
dc.date.available | 2018-09-24 | |
dc.date.copyright | 2013-09-24 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-16 | |
dc.identifier.citation | Bessler, W., Fehmel, F., Freund-Molbert, E., Knufermann, H., and Stirm, S. (1975). Escherichia coli capsule bacteriophages. IV. Free capsule depolymerase 29. J. Virol. 15, 976-984.
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The development and assessment of a bacteriocin typing method for Klebsiella. J. Hyg. 82, 207-223. Fang, C.T., Chuang, Y.P., Shun, C.T., Chang, S.C., and Wang, J.T. (2004). A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med 199, 697-705. Fung, C.P., Hu, B.S., Chang, F.Y., Lee, S.C., Kuo, B.I., Ho, M., Siu, L.K., and Liu, C.Y. (2000). A 5-year study of the seroepidemiology of Klebsiella pneumoniae: high prevalence of capsular serotype K1 in Taiwan and implication for vaccine efficacy. J Infect Dis 181, 2075-2079. Gaston, M.A., Ayling-Smith, B.A., and Pitt, T.L. (1987). New bacteriophage typing scheme for subdivision of the frequent capsular serotypes of Klebsiella spp. Journal of clinical microbiology 25, 1228-1232. Hanlon, G.W. (2007). Bacteriophages: an appraisal of their role in the treatment of bacterial infections. Int. J. Antimicrob. Agents 30, 118-128. Hughes, K.A., Sutherland, I.W., and Jones, M.V. (1998). Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase. Microbiology 144, 3039-3047. Jenney, A.W., Clements, A., Farn, J.L., Wijburg, O.L., McGlinchey, A., Spelman, D.W., Pitt, T.L., Kaufmann, M.E., Liolios, L., Moloney, M.B., et al. (2006). Seroepidemiology of Klebsiella pneumoniae in an Australian Tertiary Hospital and its implications for vaccine development. Journal of clinical microbiology 44, 102-107. Marinelli, L.J., Piuri, M., Swigonova, Z., Balachandran, A., Oldfield, L.M., van Kessel, J.C., and Hatfull, G.F. (2008). BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS One 3, e3957. Mizuta, K., Ohta, M., Mori, M., Hasegawa, T., Nakashima, I., and Kato, N. (1983). Virulence for mice of Klebsiella strains belonging to the O1 group: relationship to their capsular (K) types. Infect. Immun. 40, 56-61. Pan, Y.J., Fang, H.C., Yang, H.C., Lin, T.L., Hsieh, P.F., Tsai, F.C., Keynan, Y., and Wang, J.T. (2008). Capsular polysaccharide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype. Journal of clinical microbiology 46, 2231-2240. Pelkonen, S., Aalto, J., and Finne, J. (1992). Differential activities of bacteriophage depolymerase on bacterial polysaccharide: binding is essential but degradation is inhibitory in phage infection of K1-defective Escherichia coli. J Bacteriol 174, 7757-7761. Pieroni, P., Rennie, R.P., Ziola, B., and Deneer, H.G. (1994). The use of bacteriophages to differentiate serologically cross-reactive isolates of Klebsiella pneumoniae. J Med Microbiol 41, 423-429. Podschun, R., and Ullmann, U. (1998). Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microbiol. Rev. 11, 589-603. Rennie, R.P., and Duncan, I.B. (1974). Combined biochemical and serological typing of clinical isolates of Klebsiella. Appl. Microbiol. 28, 534-539. Rennie, R.P., Nord, C.E., Sjoberg, L., and Duncan, I.B. (1978). Comparison of bacteriophage typing, serotyping, and biotyping as aids in epidemiological surveillance of Klebsiella infections. Journal of clinical microbiology 8, 638-642. Simoliunas, E., Kaliniene, L., Truncaite, L., Klausa, V., Zajanckauskaite, A., and Meskys, R. (2012). Genome of Klebsiella sp.-infecting bacteriophage vB_KleM_RaK2. J. Virol. 86, 5406. Simoliunas, E., Kaliniene, L., Truncaite, L., Zajanckauskaite, A., Staniulis, J., Kaupinis, A., Ger, M., Valius, M., and Meskys, R. (2013). Klebsiella phage vB_KleM-RaK2 - a giant singleton virus of the family Myoviridae. PLoS One 8, e60717. Siu, L.K., Fung, C.P., Chang, F.Y., Lee, N., Yeh, K.M., Koh, T.H., and Ip, M. (2011). Molecular typing and virulence analysis of serotype K1 Klebsiella pneumoniae strains isolated from liver abscess patients and stool samples from noninfectious subjects in Hong Kong, Singapore, and Taiwan. Journal of clinical microbiology 49, 3761-3765. Taylor, L.A., and Rose, R.E. (1988). A correction in the nucleotide sequence of the Tn903 kanamycin resistance determinant in pUC4K. Nucleic Acids Res 16, 358. Tsay, R.W., Siu, L.K., Fung, C.P., and Chang, F.Y. (2002). Characteristics of bacteremia between community-acquired and nosocomial Klebsiella pneumoniae infection: risk factor for mortality and the impact of capsular serotypes as a herald for community-acquired infection. Arch. Intern. Med. 162, 1021-1027. Turton, J.F., Perry, C., Elgohari, S., and Hampton, C.V. (2010). PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets. J Med Microbiol 59, 541-547. Weinbauer, M.G. (2004). Ecology of prokaryotic viruses. FEMS Microbiol. Rev. 28, 127-181. Yurewicz, E.C., Ghalambor, M.A., Duckworth, D.H., and Heath, E.C. (1971). Catalytic and molecular properties of a phage-induced capsular polysaccharide depolymerase. J Biol Chem 246, 5607-5616. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60522 | - |
dc.description.abstract | 克雷白氏肺炎桿菌為腸內菌科的格蘭氏陰性桿菌,莢膜為重要致病因子,並且與克雷白氏肺炎桿菌所引起的感染侵襲力有關,因此正確鑑定克雷白氏肺炎桿菌之莢膜型具臨床重要意義。傳統使用抗血清分辨莢膜型,然而血清學鑑定方法的敏感度及專一性不佳,為有效進行莢膜分型,本實驗室嘗試發展噬菌體醣類分解酵素分型系統。
噬菌體感染具莢膜細菌的過程中,需要莢膜分解酵素輔助降解莢膜,而我們自原水分離的噬菌體K11-7-1-1可辨認克雷白氏肺炎桿菌丹麥標準菌種K1,K11,K21,K25,K30,K35,K64及K69共8種莢膜型,推測噬菌體K11-7-1-1應具有莢膜分解酵素,而定序其基因體序列後比對到10個與莢膜分解酵素相似之預測基因。為探討預測基因實際功能,首先以大腸桿菌蛋白質誘導系統表現蛋白質並測試活性,3個預測基因S1-1、S2-7、S2-2表現出蛋白質活性,S1-1和S2-7對應至莢膜型K11,而S2-2則對應莢膜型K25。接著以建構噬菌體基因剔除突變株方式,分析噬菌體K11-7-1-1中可能的莢膜分解酵素基因之確實功能以及莢膜型活性,得到對應至K35的S2-3及對應至K30及K69莢膜型的S2-6共兩個基因剔除株,仍有5個預測基因的功能未知。 噬菌體無標記基因剔除技術的建立,可以應用於研究噬菌體中可能為莢膜分解酵素基因,幫助確認莢膜分解酵素的活性,另外我們嘗試建造基因置換突變株,雖然目前未得到置換入外來酵素基因的突變株,但透過成功建造CRISPR之間置序列基因置換突變株,顯示基因置換株的可行性,未來可期盼基因置換株成為研究基因功能的另一方式。 | zh_TW |
dc.description.abstract | Klebsiella pnuemoniae, a Gram-negative bacillus, belongs to Enterobacteriaceae. Capsule is one of virulence factors, and researches have implied that capsular type contributes to severity of disease. Thus, how to rapidly and correctly identify the capsular type of K. pneumoniae is a clinically significant issue. Serological technique is the frequently used method in the laboratory, while further tests have to be practiced to improve the sensitivity and specificity. We attempt to establish a typing system using purified capsular depolymerases to assist diagnosis in our laboratory, and it is leading to determine the activity of putative enzyme.
During infection of encapsulated bacteria, the phages secrete endoglycosidases to degrade capsule where the primary receptor buried in order to attach the primary receptor. We isolated a new phage, named as φK11-7-1-1, which infects Denmark reference strain K1, K11, K21, K25, K30, K35, K64 as well as K69. There are 10 open reading frames (ORFs) expected to be capsular depolymerases according to sequence similarity. These putative enzymes were expressed in Escherichia coli to study the activity, S1-1 as well as S2-7 were shown to be the specific enzymes to K11, and S2-2 exhibited its activity to K25. We then constructed unmarked deletion of individual putative enzymes in φK11-7-1-1 as a mean to study functions. After construction of recombinant phages, S2-3 and S2-6 deletion mutants were obtained. S2-3 and S2-6 were separately revealed to be specific to K35, both K30 and K69. There are five putative enzymes remained unidentified function. The technique of constructing recombinant phages may be aid of and applied to study putative enzymes or gene functions in phages. We also tried to substitute a foreign enzyme gene, which ends in vain, but a replacement mutant was built by introducing a 33-nucleotide CRISPR spacer sequence, indicating that replacement mutants might be practicable as another model studying enzyme activity. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T10:20:30Z (GMT). No. of bitstreams: 1 ntu-102-R00445102-1.pdf: 2767421 bytes, checksum: 48d7bf7ec8847d1db5a44a8ea0b82be9 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 目錄
碩士學位論文口試委員會審定書....................................................................................i 致謝 ii 中文摘要 iii Abstract iv 目錄 vi 圖目錄 ix 表目錄 x 第一章、 緒論 1 1.1 噬菌體 1 1.2 噬菌體酵素與宿主感染 1 1.3 克雷白氏肺炎桿菌 2 1.4 克雷白氏肺炎桿菌之莢膜分型方法 2 1.5 噬菌體宿主範圍與所攜帶之莢膜分解酵素 4 1.6 噬菌體K11-7-1-1莢膜分解酵素可能基因之功能研究 4 第二章、 實驗材料與方法 6 2.1 實驗菌株,質體及引子 6 2.2 培養基及抗生素濃度 6 2.3 噬菌體增殖 6 2.4 純化噬菌體基因體DNA 7 2.5 聚合酶連鎖反應 8 2.6 洋菜膠電泳純化DNA片段 (agarose electrophoresis and gel extraction) 10 2.7 TA cloning 10 2.8 限制酶 (restriction enzyme digestion) 11 2.9 Blunting 11 2.10 5'端加磷酸根 (phosphate group) 12 2.11 DNA Ligation 12 2.12 載體的建構 13 2.13 純化質體 14 2.14 勝任細胞 (competent cell) 的製備 15 2.15 轉形作用 (transformation) 16 2.16 蛋白質表現與純化 17 2.17 十二烷基硫酸鈉-聚丙醯胺凝膠電泳 (SDS-PAGE) 19 2.18 西方點墨法 (Western blot) 21 2.19 同源序列區域重組 22 2.20 點試驗 (spot test) 24 2.21 噬菌體效價分析 (plaque assay) 25 2.22 噬菌體感染力分析 (infectivity assay) 25 第三章、 實驗結果 27 3.1 測試噬菌體K11-7-1-1的宿主範圍 27 3.2 定序與分析噬菌體K11-7-1-1基因體DNA序列 27 3.3 於pET-28c (+) 表現噬菌體K11-7-1-1預測蛋白質並測試其活性 28 3.4 於其他質體和大腸桿菌品系表現可能為莢膜分解酵素之基因 28 3.5 分析無活性之蛋白產物表現情形 29 3.6 噬菌體K11-7-1-1基因突變株之感染宿主選擇 29 3.7 噬菌體K11-7-1-1基因置換株 30 3.8 噬菌體K11-7-1-1之基因剔除株之宿主活性與感染力 31 第四章、 討論 34 4.1 蛋白質表現 34 4.2 噬菌體基因剔除株 34 4.3 噬菌體基因置換株 35 4.4 基因突變株之感染宿主選擇 36 4.5 醣解酵素胺基酸序列比較 36 4.6 未來應用與發展 37 第五章、 參考文獻 67 圖目錄 圖一 噬菌體K11-7-1-1定序後比對結果-Scaffold 1和Scaffold 2 39 圖二 S1-1及S2-7對K11之酵素活性 40 圖三 S2-2對K25之酵素活性 41 圖四 S1-1與S2-7胺基酸序列比較 42 圖五 基因剔除株之建造流程 43 圖六 S2-2及S2-3基因剔除株 44 圖七 ψ2-5-1-10及ψ3-8-3-2對8個莢膜型活性表現 46 圖八 ψ2-5-1-10對莢膜型K25及K35感染力分析 47 圖九 ψ2-5-1-10突變位點 48 圖十 S2-6基因剔除株 49 圖十一 ψ3-1-1對K30及K69莢膜型活性表現 50 圖十二 K30及K69醣解酵素與S2-6胺基酸序列比較 51 圖十三 S2-5基因剔除株 52 圖十四 ψ2-5-1-10,ψ3-8-3-2及ψ3-1-1對ψK11-7-1-1各莢膜型宿主感染力分析 54 圖十五 CRI-S2-3基因置換株建造流程 55 圖十六 CRI-S2-3基因置換株 56 表目錄 表一 噬菌體K11-7-1-1的10個開放閱讀框架代碼、可能對應之蛋白質產物及大小 57 表二 本研究使用的引子序列 58 表三 本研究使用之細菌菌株及質體 62 表四 本研究中λ Red recombinase之基因重組效率 64 表五 噬菌體K11-7-1-1各開放閱讀框架之蛋白質表現與功能 66 | |
dc.language.iso | zh-TW | |
dc.title | 以噬菌體重組基因技術探討多宿主噬菌體K11-7-1-1之莢膜分解酵素活性 | zh_TW |
dc.title | Determination of Capsular Depolymerase Activity in a Multihost Phage, ψK11-7-1-1, by Utilizing Recombinant Phage Techniques | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊宏志(Hung-Chih Yang),林稚容(Tzu-Lung Lin) | |
dc.subject.keyword | 莢膜噬菌體,莢膜分解酵素,重組噬菌體,基因剔除,基因置換, | zh_TW |
dc.subject.keyword | capsular bacteriophage,capsular depolymerase,recombinant phage,gene deletion,gene replacement, | en |
dc.relation.page | 70 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-16 | |
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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