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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李佳音(Chia-Yin Lee) | |
dc.contributor.author | Chieh-Wen Chang | en |
dc.contributor.author | 張絜雯 | zh_TW |
dc.date.accessioned | 2021-06-15T02:36:05Z | - |
dc.date.available | 2012-08-14 | |
dc.date.copyright | 2009-08-14 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-13 | |
dc.identifier.citation | 潘子明、王添貴、蔡金來. 1998.台灣北部地區海鮮食品腸炎弧菌之調查研究.行政院衛生署疫情報導.14:71-82.
Altarriba, M., Merino, S., Gavin, R., Canals, R., Rabaan, A., Shaw, J.G. & Tomas, J.M. 2003. A polar flagella operon (flg) of Aeromonas hydrophila contains genes required for lateral flagella expression. Microb. Pathog. 34: 249-259. Anderl, J.N., Zahller, J., Roe, F. & Stewart, P.S. 2003. Role of nutrient limitation and stationary-phase existence in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother 47:1251-1256. Ansaruzzaman, M., Lucas, M., Deen, J.L., Bhuiyan, N.A., Wang, X.Y., Safa, A., Sultana, M., Chowdhury, A., Nair, G.B., Sack, D.A., von Seidlein, L., Puri, M.K., Ali, M., Chaignat, C.L., Clemens, J.D. & Barreto, A. 2005. Pandemic serovars (O3:K6 and O4:K68) of Vibrio parahaemolyticus associated with diarrhea in Mozambique: spread of the pandemic into the African continent. J. Clin. Microbiol. 43: 2559-2562. Boles, B.R. & McCarter, L.L. 2000. Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation. J. Bacteriol 184: 5946-5954. Bowden, G.H. & Li, Y.H. 1997. Nutritional influences on biofilm development. Adv. Dent. Res. 11: 81-99. Bullock, W.O., Fernandez, J.M. & Short, J.M. 1987. Xl1-Blue: a high-efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. BioTechniques 5: 376-379. Cabello, F.C., Espejo, R.T., Hernandez, M.C., Rioseco, M.L., Ulloa, J. & Vergara, J.A. 2007. Vibrio parahaemolyticus O3:K6 epidemic diarrhea, Chile, 2005. Emerg. Infect Dis. 13: 655-656. Chen, Y.C., Chuang, Y.C., Chang, C.C., Jeang, C.L. & Chang, M.C. 2004. A K+ yptake protein, TrkA, is required for serum, protamine, and polymyxin B resistance in Vibrio vulnificus. Infect Immun 72: 629-636. Chiou, C.S., Hsu, S.Y., Chiu, S.I., Wang, T.K. & Chao, C.S. 2000. Vibrio parahaemolyticus serovar O3:K6 as cause of unusually high incidence of food-borne disease outbreaks in Taiwan from 1996 to 1999. J. Clin. Microbiol 38: 4621-4625. Clemmer, K.M. & Rather, P.N. 2008. The Lon protease regulates swarming motility and virulence gene expression in Proteus mirabilis. J. Med. Microbiol 57: 931-937. Colwell, R.R., Huq, A., Islam, M.S., Aziz, K.M., Yunus, M., Khan, N.H., Mahmud, A., Sack, R.B., Nair, G.B., Chakraborty, J., Sack, D.A. & Russek-Cohen, E. 2003. Reduction of cholera in Bangladeshi villages by simple filtration. Proc. Natl. Acad. Sci. U S A 100: 1051-1055. Connelly, M.B., Young, G.M. & Sloma, A. 2004. Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis. J. Bacteriol 186: 4159-4167. Costerton, J.W., Stewart, P.S. & Greenberg, E.P. 1999. Bacterial biofilms: a common cause of persistent infections. Science 284: 1318-1322. Daniels, N.A., MacKinnon, L., Bishop, R., Altekruse, S., Ray, B., Hammond, R.M., Thompson, S., Wilson, S., Bean, N.H., Griffin, P.M. & Slutsker, L. 2000. Vibrio parahaemolyticus infections in the United States, 1973-1998. J. Infect Dis. 181: 1661-1666. Davey, M.E. & O'Toole G, A. 2000. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol. Biol. Rev 64: 847-867. Dewanti, R. & Wong, A.C. 1995. Influence of culture conditions on biofilm formation by Escherichia coli O157:H7. Int. J. Food Microbiol 26: 147-164. Donlan, R.M. 2002. Biofilms: microbial life on surfaces. Emerg. Infect Dis. 8: 881-890. Drenkard, E. & Ausubel, F.M. 2002. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416:740-743. Enos-Berlage, J.L., Guvener, Z.T., Keenan, C.E. & McCarter, L.L. 2005. Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus. Mol. Microbiol 55: 1160-1182. Enos-Berlage, J.L. & McCarter, L.L. 2000. Relation of capsular polysaccharide production and colonial cell organization to colony morphology in Vibrio parahaemolyticus. J. Bacteriol 182: 5513-5520. Faruque, S.M., Biswas, K., Udden, S.M., Ahmad, Q.S., Sack, D.A., Nair, G.B. & Mekalanos, J.J. 2006. Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment. Proc. Natl. Acad. Sci. USA 103: 6350-6355. Ferreira, R.B., Antunes, L.C., Greenberg, E.P. & McCarter, L.L. 2008. Vibrio parahaemolyticus ScrC modulates cyclic dimeric GMP regulation of gene expression relevant to growth on surfaces. J. Bacteriol 190: 851-860. Flemming, H.C., Wingender, J., Griegbe, T. & Mayer, C. 2000. Physico-chemical properties of biofilms. In: Evans L. V., editor. Biofilms: recent advances in their study and control. Amsterdam: Harwood Academic Publishers. Fujino, T., Okino, Y., Nakada, D., Aoyama, A., Fukai, K., Mukai, T. & Ueno, T. 1953. One the bacteriological examination of shirasu food poisoning. Med. J. Osake. Univ. 4: 299-304. Fujino, T., Sakazaki, R. & Tamura, K. 1974. Designation of the type strain of Vibrio parahaemolyticus and description of 200 strains of the species. Int. J. Syst. Bacteriol. 24: 447-449. Gaddy, J.A., Tomaras, A.P. & Actis, L.A. 2009. The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells. Infect Immun 77: 3150-3160. Gulig, P.A., Bourdage, K.L. & Starks, A.M. 2005. Molecular Pathogenesis of Vibrio vulnificus. J. Microbiol 43 :118-131. Guvener, Z.T. & McCarter, L.L. 2003. Multiple regulators control capsular polysaccharide production in Vibrio parahaemolyticus. J. Bacteriol 185:5431-5441. Head, N.E. & Yu, H. 2004. Cross-sectional analysis of clinical and environmental isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity. Infect Immun 72:133-44. Honda, T. & Iida, T. 1993. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostable direct haemolysin and related haemolysin. Rev. Med. Microbiol. 4: 106-113. Honda, T., Ni, Y.X. & Miwatani, T. 1988. Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin. Infect Immun 56: 961-965. Houot, L. & Watnick, P.I. 2008. A novel role for enzyme I of the Vibrio cholerae phosphoenolpyruvate phosphotransferase system in regulation of growth in a biofilm. J Bacteriol 190:311-320. Huang, C.T., Peretti, S.W. & Bryers, J.D. 1994. Effects of medium carbon-to-nitrogen ratio on biofilm formation and plasmid stability. Biotechnol Bioeng 44: 329-336. Jaques, S. & McCarter, L.L. 2006. Three new regulators of swarming in Vibrio parahaemolyticus. J Bacteriol 188:2625-2635. Josenhans, C. & Suerbaum, S. 2002. The role of motility as a virulence factor in bacteria. Int. J. Med. Microbiol 291: 605-614. Kim, Y.K. & McCarter, L.L. 2007. ScrG, a GGDEF-EAL protein, participates in regulating swarming and sticking in Vibrio parahaemolyticus. J. Bacteriol 189:4094-4107. Kirov, S.M. 2003. Bacteria that express lateral flagella enable dissection of the multifunctional roles of flagella in pathogenesis. FEMS Microbiol Lett 224: 151-9. Kjelleberg, S., Hermansson, M., Marden, P. & Jones, G.W. 1987. The transient phase between growth and nongrowth of heterotrophic bacteria, with emphasis on the marine environment. Annu. Rev. Microbiol 41: 25-49. Lee, C.Y., Cheng, M.F., Yu, M.S. & Pan, M.J. 2002. Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus. FEMS Microbiol Lett 209: 31-37. Lim, Y., Jana, M., Luong, T.T. & Lee, C.Y. 2004. Control of glucose- and NaCl-induced biofilm formation by rbf in Staphylococcus aureus. J. Bacteriol 186:722-729. Makino, K., Oshima, K., Kurokawa, K., Yokoyama, K., Uda, T., Tagomori, K., Iijima, Y., Najima, M., Nakano, M., Yamashita, A., Kubota, Y., Kimura, S., Yasunaga, T., Honda, T., Shinagawa, H., Hattori, M. & Iida, T. 2003. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae. Lancet 361:743-749. Martinez-Urtaza, J., Simental, L., Velasco, D., DePaola, A., Ishibashi, M., Nakaguchi, Y., Nishibuchi, M., Carrera-Flores, D., Rey-Alvarez, C. & Pousa, A. 2005. Pandemic Vibrio parahaemolyticus O3:K6, Europe. Emerg. Infect Dis. 11: 1319-1320. McCarter, L., Hilmen, M. & Silverman, M. 1988. Flagellar dynamometer controls swarmer cell differentiation of V. parahaemolyticus. Cell 54: 345-351. McCarter, L.L. 1998. OpaR, a homolog of Vibrio harveyi LuxR, controls opacity of Vibrio parahaemolyticus. J. Bacteriol 180:3166-3173. McCarter, L.L. 2001. Polar flagellar motility of the Vibrionaceae. Microbiol Mol. Biol. Rev 65:445-462, table of contents. Merino, S., Shaw, J.G. & Tomas, J.M. 2006. Bacterial lateral flagella: an inducible flagella system. FEMS Microbiol Lett 263: 127-135. Miyamoto, Y., Obara, Y., Nikkawa, T., Yamai, S., Kato, T., Yamada, Y. & Ohashi, M. 1980. Simplified purification and biophysicochemical characteristics of Kanagawa phenomenon-associated hemolysin of Vibrio parahaemolyticus. Infect Immun. 28: 567-576. Nair, G.B., Ramamurthy, T., Bhattacharya, S.K., Dutta, B., Takeda, Y. & Sack, D.A. 2007. Global dissemination of Vibrio parahaemolyticus serotype O3:K6 and its serovariants. Clin. Microbiol Rev. 20: 39-48. Nomura, T., Hamashima, H. & Okamoto, K. 2000. Carboxy terminal region of haemolysin of Aeromonas sobria triggers dimerization. Microb. Pathog. 28:25-36. O'Toole, G., Kaplan, H.B. & Kolter, R. 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol 54: 49-79. O'Toole, G.A., Gibbs, K.A., Hager, P.W., Phibbs, P.V., Jr. & Kolter, R. 2000. The global carbon metabolism regulator Crc is a component of a signal transduction pathway required for biofilm development by Pseudomonas aeruginosa. J. Bacteriol 182: 425-431. Okuda, J., Ishibashi, M., Hayakawa, E., Nishino, T., Takeda, Y., Mukhopadhyay, A.K., Garg, S., Bhattacharya, S.K., Nair, G.B. & Nishibuchi, M. 1997. Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan. J. Clin. Microbiol 35: 3150-3155. Ottaviani, D., Leoni, F., Rocchegiani, E., Santarelli, S., Canonico, C., Masini, L., Ditrani, V. & Carraturo, A. 2008. First clinical report of pandemic Vibrio parahaemolyticus O3:K6 infection in Italy. J. Clin. Microbiol 46: 2144-2145. Overhage, J., Bains, M., Brazas, M.D. & Hancock, R.E. 2008. Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J. Bacteriol 190: 2671-2679. Park, K.S., Arita, M., Iida, T. & Honda, T. 2005. vpaH, a gene encoding a novel histone-like nucleoid structure-like protein that was possibly horizontally acquired, regulates the biogenesis of lateral flagella in trh-positive Vibrio parahaemolyticus TH3996. Infect Immun 73: 5754-5761. Park, K.S., Ono, T., Rokuda, M., Jang, M.H., Iida, T. & Honda, T. 2004. Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus. Microbiol Immunol 48:313-318. Philippe, N., Alcaraz, J.P., Coursange, E., Geiselmann, J. & Schneider, D. 2004. Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid 51:246-255. Picioreanu, C., van Loosdrecht, M.C. & Heijnen, J.J. 2000. A theoretical study on the effect of surface roughness on mass transport and transformation in biofilms. Biotechnol Bioeng 68: 355-369. Rashid, M.H., Rajanna, C., Zhang, D., Pasquale, V., Magder, L.S., Ali, A., Dumontet, S. & Karaolis, D.K. 2004. Role of exopolysaccharide, the rugose phenotype and VpsR in the pathogenesis of epidemic Vibrio cholerae. FEMS Microbiol Lett 230:105-13. Rui, H., Liu, Q., Wang, Q., Ma, Y., Liu, H., Shi, C. & Zhang, Y. 2009. Role of Alkaline Serine Protease, Asp, in Vibrio alginolyticus Virulence and Regulation of Its Expression by LuxO-LuxR Regulatory System. J. Microbiol Biotechnol 19: 431-438. Sakazaki, R., Iwanami, S. & Fukumi, H. 1963. Studies on the enteropathogenic, facultatively halophilic bacteria, Vibrio parahaemolyticus. I. Morphological, cultural and biochemical properties and its taxonomical position. Jpn. J. Med. Sci. Biol. 16: 161-188. Sarkar, B.L., Kumar, R., De, S.P. & Pal, S.C. 1987. Hemolytic activity of and lethal toxin production by environmental strains of Vibrio parahaemolyticus. Appl. Environ. Microbiol 53: 2696-2698. Savoia, D. & Zucca, M. 2007. Clinical and environmental Burkholderia strains: biofilm production and intracellular survival. Curr. Microbiol 54: 440-444. Schafer, A., Kalinowski, J., Simon, R., Seep-Feldhaus, A.H. & Puhler, A. 1990. High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria. J. Bacteriol 172: 1663-1666. Shikuma, N.J. & Yildiz, F.H. 2009. Identification and characterization of OscR, a transcriptional regulator involved in osmolarity adaptation in Vibrio cholerae. J. Bacteriol 191: 4082-4096. Shime-Hattori, A., Iida, T., Arita, M., Park, K.S., Kodama, T. & Honda, T. 2006. Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation. FEMS Microbiol Lett 264: 89-97. Spratt, D.A., Latif, J., Montebugnoli, L.L. & Wilson, M. 2004. In vitro modeling of dental water line contamination and decontamination. FEMS Microbiol Lett 235: 363-367. Stewart, B.J., Enos-Berlage, J.L. & McCarter, L.L. 1997. The lonS gene regulates swarmer cell differentiation of Vibrio parahaemolyticus. J .Bacteriol 179: 107-114. Stewart, B.J. & McCarter, L.L. 2003. Lateral flagellar gene system of Vibrio parahaemolyticus. J. Bacteriol 185:4508-4518. Stoodley, P., Sauer, K., Davies, D.G. & Costerton, J.W. 2002. Biofilms as complex differentiated communities. Annu. Rev. Microbiol 56: 187-209. Takeda, Y., Takeda, T., Honda, T. & Miwatani, T. 1978. Comparison of bacterial cardiotoxins: thermostable direct hemolysin from Vibrio parahaemolyticus, streptolysin O and hemolysin from Listeria monocytogenes. Biken J. 21: 1-8. Wang, J., Sasaki, T., Maehara, Y., Nakao, H., Tsuchiya, T. & Miyoshi, S. 2008 Variation of extracellular proteases produced by Vibrio vulnificus clinical isolates: genetic diversity of the metalloprotease gene (vvp), and serine protease secretion by vvp-negative strains. Microb Pathog 44: 494-500. Yamaichi, Y., Iida, T., Park, K.S., Yamamoto, K. & Honda, T. 1999. Physical and genetic map of the genome of Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species. Mol Microbiol 31:1513-1521. Yamazaki, M., Inuzuka, K., Matsumoto, M., Miwa, Y., Hiramatsu, R., Matsui, H., Sakae, K., Suzuki, Y. & Miyazaki, Y. 2003. [Epidemiological study of outbreaks and sporadic cases due to Vibrio parahaemolyticus--serotype O3:K6 in Aichi Prefecture, Japan, during 1988 and 2001]. Kansenshogaku Zasshi 77:1015-1023. Yildiz, F.H. & Visick, L.K. 2008. Vibrio biofilms: so much the same yet so different. Trends in Microbiology 17: 109-118 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44008 | - |
dc.description.abstract | 前人研究(FEMS Microbiology Letter (2002) 209:31-37)已證實胞外絲胺酸蛋白酶PrtA可能為一毒力因子。已有報告指出,胞外蛋白酶會參與革蘭氏陽性菌的遊走能力。此外,遊走能力與生物膜的形成及致病能力有關,故本研究擬探討PrtA對於腸炎弧菌生物膜的形成及遊走能力扮演的角色。由於腸炎弧菌O3:K6菌株為我國與全球性食物中毒致病菌,本研究選擇三株腸炎弧菌O3:K6臨床菌株、一株type strain ATCC17802以及五株分離自台灣的環境菌株為研究材料,並建構O3:K6和type strain的prtA缺陷株和其補償株。首先利用接合生殖將帶有prtA缺失片段的自殺質體pDS132-mprtA送入腸炎弧菌中,篩選得到prtA缺陷株,再利用核酸序列定序、南方漬片和西方漬片進行確認。接著將帶有prtA的質體pSA19CP-MCS-prtA以電衝方式送入prtA缺陷株中製備補償株。在含3%NaCl之tryptic soy broth (TSB-3% NaCl)或是含5%NaCl之marine broth (MB-5% NaCl),37 ℃的培養條件下觀察其生長情形,O3:K6和type strain的野生株、缺陷株和補償株的生長情形皆無顯著差異。將腸炎弧菌培養在37 ℃,TSB-3% NaCl和MB-5% NaCl兩種培養基中,測定胞外蛋白酶的活性,結果發現類似海洋環境的Marine broth才能誘導膠原蛋白酶產生,並有利於生物膜的形成。另在TSB-3% NaCl,37 ℃下培養,O3:K6菌株prtA缺陷株的生物膜形成能力明顯野生株增強,顯示prtA缺失能使腸炎弧菌在TSB-3% NaCl的環境中增加生物膜形成能力。但在MB-5% NaCl,30 ℃的條件下培養,腸炎弧菌ATCC17802的生物膜生成是需要prtA的參與,prtA缺失會造成生物膜生成量明顯降低。但相同的條件對O3:K6菌株而言,prtA缺失不會造成生物膜生成量的改變。藉由含0.25% agar的TSA-3%NaCl來測定菌株的遊走能力,結果發現prtA缺陷株的菌落大小比野生株明顯較小,顯示prtA缺失會造成遊走能力的降低。而補償株則生物膜生成能力與遊走能力則介於野生株和缺陷株之間。比較環境菌株以及臨床菌株,發現大部份環境菌株(4/5)生物膜的生成能力比臨床菌株強,但環境菌株整體的遊走能力則與臨床菌株無太大差異。當TSB培養基鹽濃度含1%和3%時,生物膜的生成量最高,而在MB培養基中,鹽濃度對於生物膜生成量則無明顯影響。在不同的條件培養下,由於不同的營養成分造成生物膜的生合成能力有所差異,而prtA不僅與致病能力有關,同時也和菌體在環境中的生存能力有關。 | zh_TW |
dc.description.abstract | Protease A, serine protease of Vibrio parahaemolyticus, has been known to possess cytotoxicity and considered as a putative virulence factor. We constructed the prtA deletion mutants and their complement strains with three O3:K6 serotype strains and one V. parahaemolyticus type strain, ATCC17802. Campared the wild-type, prtA deletion mutants and their complement strains, their growth rates was not significant difference in TSB3 or MB5 medium at 37 ℃. But only strains cultured in MB5 medium, it could induce much protease activities from wild-type and a little protease activities from prtA deletion mutants. When comparing the biofilm formation between the wild type and the prtA deletion mutants, the prtA deletion mutants increased biofilm formation in TSB3 at 37 ℃ significantly. The mutants lacking prtA significantly reduced swarming motility on TSB3 semisolid agar at both 37 ℃ and 30 ℃. The type strain and most environmental strains had much more biofilm formation than those of clinical strains in MB5 medium at both 37 ℃ and 30 ℃. However, only prtA deletion mutant of type strain VP00 reduced half of biofilm formation on MB5 medium at 30 ℃ was observed. In different saline of TSB medium, the TSB1 and TSB3 medium induced most biofilm formation from V. parahaemolyticus strains. But no specific saline concentration of MB medium could induced most biofilm formation.These results indicated that prtA is growth independent and significantly affect the biofilm formation in TSB3 medium at 37 ℃ and the swarming motilities of V. parahaemolyticus. We conclude that when cultured at different temperatures and medium, PrtA leads to different microbial physiology and metabolism. The regulation of biofilm formation affected by culture temperature and medium slightly in O3:K6 strains but obviously in environmental strains from marine sources. Our results indicate that the regulation of biofilm formation in O3:K6 and in environmental strains had different. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T02:36:05Z (GMT). No. of bitstreams: 1 ntu-98-R96623023-1.pdf: 7519630 bytes, checksum: 232771a7a800d1f2a371b9713a1950a8 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 目錄
口試委員會審定書 i 謝誌 ii 中文摘要 iv 英文摘要 vi 目錄 viii 表次 xii 圖次 xiii 壹、前言 1 一、 腸炎弧菌 1 1. 概論 1 2. 腸炎弧菌疾病流行情形 2 3. 腸炎弧菌基因體 3 4. 毒力因子 4 5. 絲胺酸蛋白酶A 5 二、 生物膜(biofilm) 6 1. 生物膜的定義 6 2. 生物膜之形成 6 3. 生物膜之結構 7 4. 弧菌與生物膜 8 5. 腸炎弧菌生物膜生合成相關基因 9 三、 側鞭毛系統 10 1. 鞭毛與菌體移動 10 2. 側鞭毛基因 11 3. 側鞭毛調控系統 11 四、 研究目的與緣起 12 貳、實驗材料與方法 14 I. 實驗材料 14 一、 實驗菌株、質體與引子 14 二、 培養基 14 三、 藥品與試劑 14 四、 蛋白質膠體電泳之緩衝液與試劑 15 五、 西方漬片所需之溶液與試劑 16 六、 實驗使用套組 17 II. 實驗方法 18 一、 一般DNA技術 18 1. DNA瓊脂膠體電泳 18 2. 染色體DNA製備 18 3. 質體製備 19 4. 聚合酶連鎖反應(polymerase chain reaction, PCR) 19 5. DNA片段純化 19 6. 接合反應(Ligation) 20 7. 製備勝任細胞(Competent cell) 20 (1) 大腸桿菌 (E.coli) 20 (2) 腸炎弧菌 (V. parahaemolyticus) 20 8. 電衝法轉型作用 21 9. 南方漬片(Southern blotting) 21 二、 一般蛋白質技術 23 1. 蛋白質濃度測定 23 2. SDS-PAGE蛋白質膠體電泳 23 3. 西方漬片(Western blotting) 23 三、 建構腸炎弧菌prtA缺陷株 24 1. 建構mutant prtA fragment 24 2. 將prtA缺失片段和pDS132(suicide vector)連接 25 3. 以接合生殖將帶有prtA缺失片段之質體pDS132-mprtA,送入腸炎弧菌中 25 四、 建構腸炎弧菌prtA補償株 26 五、 腸炎弧菌野生株、prtA缺陷株與補償株基本生理測定 27 1. 生長曲線測定 27 2. 膠原蛋白酶活性測定 27 六、 腸炎弧菌生物膜生合成測定 28 七、 腸炎弧菌遊走能力測定 28 八、 統計分析 29 参、 實驗結果 30 一、 腸炎弧菌O3:K6臨床株和環境菌株prtA之序列比對 30 二、 建構腸炎弧菌prtA缺陷株 31 三、 建構腸炎弧菌prtA缺陷株之補償株 33 四、 腸炎弧菌野生株、prtA株與補償株生長速率與胞外蛋白酶表現 34 五、 腸炎弧菌環境菌株與臨床菌株生物膜生成之差異 35 六、 腸炎弧菌環境菌株與臨床菌株遊走能力之差異 36 七、 腸炎弧菌野生株、prtA缺陷株與補償株於不同條件下之生物膜形成的比較 36 八、 腸炎弧菌野生株、prtA缺陷株與補償株之遊走現象的比較 37 九、 鹽濃度對生物膜生成的影響 37 肆、討論 39 一、 腸炎弧菌蛋白酶A與生物膜的生合成之相關性 39 二、 環境菌株與臨床菌株形成生物膜生合成之差異 40 三、 培養條件對生物膜生合成量之影響 40 四、 腸炎弧菌蛋白酶A與其遊走現象之相關性 42 五、 未來發展方向 42 伍、結論 43 陸、參考文獻 45 柒、表 59 捌、圖 70 玖、附錄表 105 表次 表一、本研究所使用的菌株與質體 59 表二、本研究所使用的引子 61 表三、不同的腸炎弧菌菌株在37 ℃於TSB3或MB5之生長速率 62 表四、腸炎弧菌臨床菌株和環境菌株在不同條件下生物膜生成量 63 表五、腸炎弧菌臨床菌株和環境菌株在不同溫度下遊走能力的比較 64 表六、不同腸炎弧菌在不同條件下生物膜生成量 65 表七、不同腸炎弧菌在不同條件下遊走能力的比較 66 表八、腸炎弧菌type strain於不同鹽濃度生物膜生合成比較 67 表九、腸炎弧菌O3:K6 VP1593於不同鹽濃度生物膜生合成比較 68 表十、腸炎弧菌環境菌株於不同鹽濃度生物膜生合成比較 69 圖次 圖一、不同腸炎弧菌菌株的prtA核苷酸序列與VPA0227核苷酸序列比對 70 圖二、腸炎弧菌O3:K6之VP1593之prtA胺基酸序列與VPA0227胺基酸序列比對 77 圖三、建構prtA缺陷株之原理 79 圖四、利用基因定序確認prtA缺陷株之序列 80 圖五、利用南方漬片分析腸炎弧菌野生株、缺陷株與補償株之染色體和質體DNA 82 圖六、腸炎弧菌野生株、缺陷株與補償株之胞外蛋白質之SDS-PAGE與西方漬片 85 圖七、腸炎弧菌O3:K6野生株、prtA缺陷株與補償株於TSB3之生長情形 87 圖八、腸炎弧菌VP00、prtA缺陷株與補償株於不同培養基之生長情形 88 圖九、於不同培養基腸炎弧菌野生株與prtA缺陷株培養之膠原蛋白酶活性 89 圖十、比較不同培養條件下臨床菌株與環境菌株生物膜生成量 90 圖十一、比較不同溫度條件下臨床菌株與環境菌株遊走能力 91 圖十二、比較腸炎弧菌野生株、prtA缺陷株與補償株於不同條件下之生物膜形成 92 圖十三、於37 ℃和30 ℃下,在TSA3-0.25% agar上腸炎弧菌野生株、prtA缺陷株與補償株之菌落大小 95 圖十四、在不同溫度下比較腸炎弧菌野生株、prtA缺陷株與補償株之遊走現象 96 圖十五、比較腸炎弧菌VP00野生株、prtA缺陷株與補償株於不同鹽濃度下之生物膜形成 99 圖十六、比較腸炎弧菌VP1593野生株、prtA缺陷株與補償株於不同鹽濃度下之生物膜形成 101 圖十七、比較腸炎弧菌環境菌株VP02和VP03不同鹽濃度下之生物膜形成 103 | |
dc.language.iso | zh-TW | |
dc.title | 胞外絲胺酸蛋白酶對腸炎弧菌生物膜生成及遊走能力之影響 | zh_TW |
dc.title | The Extracellular Serine Protease Affects the Biofilm Formation and Swarming Motility in Vibrio parahaemolyticus | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 邱乾順,余美萱,彭雲明 | |
dc.subject.keyword | 腸炎弧菌,絲胺酸蛋白酶,生物膜,遊走能力, | zh_TW |
dc.subject.keyword | Vibrio parahaemolyticus,serine protease,biofilm,swarming motility, | en |
dc.relation.page | 105 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-08-13 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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