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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王錦堂 | |
dc.contributor.author | Kai-chiang Tung | en |
dc.contributor.author | 董凱強 | zh_TW |
dc.date.accessioned | 2021-06-13T00:01:08Z | - |
dc.date.available | 2012-08-08 | |
dc.date.copyright | 2007-08-08 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-30 | |
dc.identifier.citation | 1. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998;11:589-603
2. Sahly H, Podschun R. Clinical, bacteriological, and serological aspects of Klebsiella infections and their spondylarthropathic sequelae. Clin Diagn Lab Immunol 1997;4:393-9 3. Chang SC, Fang CT, Hsueh PR, Chen YC and Luh KT. Klebsiella pneumoniae isolates causing liver abscess in Taiwan. Diagn Microbiol Infect Dis 2000;37:279-84 4. Fang CT, Chang SC, Hsueh PR, Chen YC, Sau WY and Luh KT. Microbiologic features of adult community-acquired bacterial meningitis in Taiwan. J Formos Med Assoc 2000;99:300-4 5. Fung CP, Chang FY, Lee SC, et al. A global emerging disease of Klebsiella pneumoniae liver abscess: is serotype K1 an important factor for complicated endophthalmitis? Gut 2002;50:420-4 6. Cheng DL, Liu YC, Yen MY, Liu CY and Wang RS. Septic metastatic lesions of pyogenic liver abscess. Their association with Klebsiella pneumoniae bacteremia in diabetic patients. Arch Intern Med 1991;151:1557-9 7. Chiu CT, Lin DY and Liaw YF. Metastatic septic endophthalmitis in pyogenic liver abscess. J Clin Gastroenterol 1988;10:524-7 8. Cheng DL, Liu YC, Yen MY, Liu CY, Shi FW and Wang LS. Pyogenic liver abscess: clinical manifestations and value of percutaneous catheter drainage treatment. J Formos Med Assoc 1990;89:571-6 9. Fang CT, Chen YC, Chang SC, Sau WY and Luh KT. Klebsiella pneumoniae meningitis: timing of antimicrobial therapy and prognosis. Qjm 2000;93:45-53 10. Lu CH, Chang WN, Chuang YC and Chang HW. The prognostic factors of adult gram-negative bacillary meningitis. J Hosp Infect 1998;40:27-34 11. Jang TN, Wang FD, Wang LS, Yu KW and Liu CY. Gram-negative bacillary meningitis in adults: a recent six-year experience. J Formos Med Assoc 1993;92:540-6 12. Fang CT, Chuang YP, Shun CT, Chang SC and Wang JT. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med 2004;199:697-705 13. Russel M, Linderoth NA and Sali A. Filamentous phage assembly: variation on a protein export theme. Gene 1997;192:23-32 14. Bernhardt TG, Struck DK and Young R. The lysis protein E of phi X174 is a specific inhibitor of the MraY-catalyzed step in peptidoglycan synthesis. J Biol Chem 2001;276:6093-7 15. Bernhardt TG, Wang IN, Struck DK and Young R. A protein antibiotic in the phage Qbeta virion: diversity in lysis targets. Science 2001;292:2326-9 16. Bernhardt TG, Roof WD and Young R. Genetic evidence that the bacteriophage phi X174 lysis protein inhibits cell wall synthesis. Proc Natl Acad Sci U S A 2000;97:4297-302 17. Young I, Wang I and Roof WD. Phages will out: strategies of host cell lysis. Trends Microbiol 2000;8:120-8 18. Takac M, Witte A and Blasi U. Functional analysis of the lysis genes of Staphylococcus aureus phage P68 in Escherichia coli. Microbiology 2005;151:2331-42 19. Ziedaite G, Daugelavicius R, Bamford JK and Bamford DH. The Holin protein of bacteriophage PRD1 forms a pore for small-molecule and endolysin translocation. J Bacteriol 2005;187:5397-405 20. Bernhardt TG, Wang IN, Struck DK and Young R. Breaking free: 'protein antibiotics' and phage lysis. Res Microbiol 2002;153:493-501 21. Loessner MJ. Bacteriophage endolysins--current state of research and applications. Curr Opin Microbiol 2005;8:480-7 22. Borysowski J, Weber-Dabrowska B and Gorski A. Bacteriophage endolysins as a novel class of antibacterial agents. Exp Biol Med (Maywood) 2006;231:366-77 23. Pritchard DG, Dong S, Baker JR and Engler JA. The bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30. Microbiology 2004;150:2079-87 24. Cheng Q, Nelson D, Zhu S and Fischetti VA. Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme. Antimicrob Agents Chemother 2005;49:111-7 25. Navarre WW, Ton-That H, Faull KF and Schneewind O. Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity. J Biol Chem 1999;274:15847-56 26. Yokoi KJ, Kawahigashi N, Uchida M, et al. The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage varphiWMY: cloning, sequencing, expression, and mutational analysis in Escherichia coli. Gene 2005;351:97-108 27. Wang IN, Deaton J and Young R. Sizing the holin lesion with an endolysin-beta-galactosidase fusion. J Bacteriol 2003;185:779-87 28. Low LY, Yang C, Perego M, Osterman A and Liddington RC. Structure and lytic activity of a Bacillus anthracis prophage endolysin. J Biol Chem 2005;280:35433-9 29. Orito Y, Morita M, Hori K, Unno H and Tanji Y. Bacillus amyloliquefaciens phage endolysin can enhance permeability of Pseudomonas aeruginosa outer membrane and induce cell lysis. Appl Microbiol Biotechnol 2004;65:105-9 30. Zimmer M, Vukov N, Scherer S and Loessner MJ. The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains. Appl Environ Microbiol 2002;68:5311-7 31. Loeffler JM, Nelson D and Fischetti VA. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 2001;294:2170-2 32. Yoong P, Schuch R, Nelson D and Fischetti VA. Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. J Bacteriol 2004;186:4808-12 33. Paradis-Bleau C, Cloutier I, Lemieux L, et al. Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage phiKZ gp144 lytic transglycosylase. FEMS Microbiol Lett 2007;266:201-9 34. Sass P, Bierbaum G. Lytic activity of recombinant bacteriophage phi11 and phi12 endolysins on whole cells and biofilms of Staphylococcus aureus. Appl Environ Microbiol 2007;73:347-52 35. Pellegrini A, Thomas U, von Fellenberg R and Wild P. Bactericidal activities of lysozyme and aprotinin against gram-negative and gram-positive bacteria related to their basic character. J Appl Bacteriol 1992;72:180-7 36. Ibrahim HR, Yamada M, Matsushita K, Kobayashi K and Kato A. Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus. J Biol Chem 1994;269:5059-63 37. During K, Porsch P, Mahn A, Brinkmann O and Gieffers W. The non-enzymatic microbicidal activity of lysozymes. FEBS Lett 1999;449:93-100 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28107 | - |
dc.description.abstract | 比對克雷伯氏肺炎桿菌(Klebsiella pneumoniae) NTUH-K2044菌株與MGH 78578菌株之DNA序列,發現在NTUH-K2044全基因體序列之2089556至2114933鹼基對(base pairs)位置(約25 kb)與MGH 78578菌株之序列有很大的差異。以National center for biotechnology information Basic Local Alignment Search Tool (NCBI BLAST)生物資訊工具分析此段序列,發現其中有一開放讀架(open reading frame)預測為溶菌酶(endolysin)基因。溶菌酶為噬菌體(phage)進行溶菌期(lytic cycle)所需之酵素,其能作用在細菌之肽聚醣(peptidoglycan)而使細菌之細胞壁分解,以利組裝好的噬菌體釋出。先前的研究報告指出,使用外加溶菌酶的方式作用於細菌,能夠使細菌細胞壁分解而殺死之,這也使得溶菌酶具有作為替代性殺菌劑的發展潛力。本實驗將此預測之溶菌酶基因轉殖於大腸桿菌(Escherichia coli),進一步表現與純化此蛋白質。將此蛋白質作用於不同細菌,測試作用不同時間下的細菌存活率。結果顯示,以蛋白質50微克(μg)作用於大腸桿菌15分鐘,即能降低細菌存活率至7.22 %。另外,此蛋白質的殺菌效力具有選擇性,受測菌株中,對於大腸桿菌的殺菌效力最佳,其次為克雷伯氏肺炎桿菌,對於枯草桿菌(Bacillus subtilis)效果較差。為了更進一步確認此蛋白質作用的受質為肽聚醣,因此將蛋白質加入肽聚醣混濁液進行肽聚醣分解實驗(peptidoglycan degrading assay),結果發現此蛋白質無法對枯草桿菌之肽聚醣進行分解。推測此蛋白質的殺菌效果,可能是針對細菌其他的部分作為標的。 | zh_TW |
dc.description.abstract | Comparison of the genome sequences of Klebsiella pneumoniae NTUH-K2044 strain with those of MGH 78578 strain, we found that there is a different region (about 25 kb) in the 2089556 to 2114933 sites of the genome of NTUH-K2044 strain. Analysis of the different region by using bioinformatics tools, National center for biotechnology information Basic Local Alignment Search Tool (NCBI BLAST), we found that there is an open reading frame being predicted as an endolysin gene. Endolysin is the enzyme encoded by bacteriophage and directly targets bonds in the peptidoglycan layer of the bacterial cell wall. The result of this activity is degradation of the rigid murein layer and release of newly assembled virions at the terminal stage of the phage reproduction cycle. According to previous reports, the capability of endolysin to digest the cell wall when applying exogenously to bacterial cells has enabled its use as alternative antibacterials. The putative endolysin gene was clone to Escherichia coli, and protein was expressed and purificated. And then we applied the purified protein exogenously to bacterial cells, and their survival rate was observed. The results showed that 50 μg purified protein can decrease the survival rate of Escherichia coli to 7.22% in 15 minutes. On the other hand, the protein also showed selective bactericidal activity. Among the tested strains, Escherichia coli is the most sensitive to the protein, Klebsiella pneumoniae is the second, and Bacillus subtilis is the least sensitive to the putative endolysin. In order to find out if peptidoglycan is the substrate of the protein, we performed peptidoglycan degrading assay. The results showed that the protein can not degrade peptidoglycan of Bacillus subtilis. It may be other components of bacteria that the protein targets, and results in the bactericidal effect. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T00:01:08Z (GMT). No. of bitstreams: 1 ntu-96-R94445104-1.pdf: 1167757 bytes, checksum: 6e9c6988e79f218301c7c5cea4cf192a (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 中文摘要 …………………………………………………………… ΙII
Abstract ………………………………………………………………IV 目錄 ……………………………………………………………………VI 圖目錄 ………………………………………………………………VIII 表目錄 …………………………………………………………………IX 第一章、緒論 1.克雷伯氏肺炎桿菌 …………………………………………………… 1 2.溶菌酶 ………………………………………………………………… 2 3.研究目的 ……………………………………………………………… 5 第二章、材料及方法 1.菌株 …………………………………………………………………… 6 2.質體及培養基 ………………………………………………………… 7 3.克雷伯氏肺炎桿菌NTUH-K2044菌株與MGH 78578菌株之 生物資訊分析……………………………………………………………8 4.克雷伯氏肺炎桿菌染色體 DNA 的萃取 ……………………… 8 5. 預測之溶菌酶基因KP2177表現載體的建構 ………………… 9 6. His 標誌 KP2177 蛋白質的表現與分析 10 7. 大量純化 His 標誌 KP2177 蛋白質 ……………………… 12 8. 純化之 His 標誌 KP2177 蛋白質分析 …………………… 13 9. KP2177蛋白質對細菌存活率的影響 ……………………… 14 10. 肽聚醣分解實驗 …………………………………………………… 15 第三章、結果 1.克雷伯氏肺炎桿菌生資訊分析…………………………17 2.His 標誌 KP2177 蛋白質的表現與分析 ……………… 18 3. KP2177蛋白質對細菌存活率的影響 …………………… 19 4.肽聚醣分解實驗 …………………………………………… 20 第四章、討論 ……………………………………………………………… 21 第五章、參考文獻 …………………………………………………………… 38 圖目錄 圖一、預測溶菌酶基因KP2177表現載體的建構……………………………… 25 圖二、 Coomassie blue 全細胞蛋白質染色 ……………………………………26 圖三、純化之His標誌KP2177蛋白質 Coomassie blue 染色 ……………… 27 圖四、純化之His標誌KP2177蛋白質西方點漬法(Western blot)…………28 圖五、純化之蛋白質KP2177對大腸桿菌(E. coli)細菌存活率實驗……………29 圖六、純化之蛋白質KP2177對克雷伯氏肺炎桿菌(K. pneumoniae)細菌存活率實驗……………………………………………… 30 圖七、純化之蛋白質KP2177對枯草桿菌(B. subtilis)細菌存活率實驗--------------31 圖八、肽聚醣分解實驗(peptidoglycan degrading assay)……………………… 32 表目錄: 表一、克雷伯氏肺炎桿菌NTUH-K2044菌株全基因體序列之2089556 至2114933鹼基對位置類似於噬菌體之基因………………………… 33 表二、 KP2177之 DNA序列及胺基酸序列 ………………………………… 34 表三、 KP2176之 DNA序列及胺基酸序列……………………………………35 表四、利用生物資訊工具-National center for biotechnology information Basic Local Alignment Search Tool (NCBI BLAST)之胺基酸序列資料庫,分析比對KP2177 胺基酸序列之結果………………………………………………36 表五、利用生物資訊工具-National center for biotechnology information Basic Local Alignment Search Tool (NCBI BLAST)之胺基酸序列資料庫,分析比對KP2176 胺基酸序列之結果………………………………………………37 | |
dc.language.iso | zh-TW | |
dc.title | 克雷伯氏肺炎桿菌預測溶菌酶之選殖與功能分析 | zh_TW |
dc.title | Cloning and functional characterization of a putative endolysin in Klebsiella pneumoniae | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧麗珍,陳美如 | |
dc.subject.keyword | 溶菌酶,克雷伯氏肺炎桿菌,殺菌,肽,聚醣,噬菌體, | zh_TW |
dc.subject.keyword | endolysin,Klebsiella pneumoniae,bactericidal effect,peptidoglycan,phage, | en |
dc.relation.page | 43 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-31 | |
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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