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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 | Hui-Chih Fan | en |
dc.contributor.author | 范惠芝 | zh_TW |
dc.date.accessioned | 2021-06-13T08:12:44Z | - |
dc.date.available | 2006-08-02 | |
dc.date.copyright | 2005-08-02 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-20 | |
dc.identifier.citation | Alm RA, Ling LS, Moir DT, King BL Brown ED, Doig PC, Smith DR, Noonan B, Guild BC, Dejonge BL, Carmel G, Tummino PJ, Caruso A, Uria-Nickelsen M, Mills DM, Ives C, Gibson R, Merberg D, Mills SD, Jiang Q, Taylor DE, Vovis GF, Trust TJ. Genomic-seguence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. (1999) Nature 397:176-180
Ang S, Lee CZ, Peck K, Sindici M, Udayakumar Matrubutham, Gleeson MA, Wang JT. Acid-induced gene expression in Helicobacter pylori:study in genomic scale by microarray. (2001) Infect Iimmun. 69(3):1679-1686 Bernhardt TG, de Boer PA. Screening for synthetic lethal mutants in Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with murein hydrolase activity. (2004) Mol Microbiol. 52(5) 1255-1269 Bijlsma JJ, Lie-A-Ling M, Nootenboom IC, Vandenbroucke-Grauls CM, Kusters JG. Identification of loci essential for the growth of Helicobacter pylori under acidic conditions. (2000) J Infect Dis. 182(5):1566-9 Blaser MJ. Helicobacter pylori: Its role in disease. (1992) Clin Infect Dis. 15:386-391 Blaser MJ. Helicobacter pylori: microbiology of a 'slow' bacterial infection. (1993) Trends Microbiol. 1(7):255-260 Blaser MJ, Atherton JC. Helicobacter pylori persistence: biology and disease. (2004) J Clin Invest. 113(3):321-333 Boren T, Falk P, Roth KA, Larson G, Normark S. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. (1993) Science 262(5141):1892-1895 Bumann D, Aksu S, Wendland M, Janek K, Zimny-Arndt U, Sabarth N, Meyer TF, Jungblut. Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori. (2002) Infect Immun. 70(7): 3396-3403 Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, Rappuoli R, Covacci A. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. (1996) Proc Natl Acad Sci U S A. 93 (25):14648-14653 Cortes G, de Astorza B, Benedi VJ, Alberti S. Role of the htrA gene in Klebsiella pneumoniae virulence. (2002) Infect Immun. 70(9):4772-4776 Covacci A, Telford JL, Giudice GD, Parsonnet J, Rappuoli. Helicobascter pylori virulence and genetic geography (1999) Science 284:1328-1333 de Bernard M, Papini E, de Filippis V, Gottardi E, Telford J, Manetti R, Fontana A, Rappuoli R, Montecucco C. Low pH activates the vacuolating toxin of Helicobacter pylori, which becomes acid and pepsin resistant. (1995) J Biol Chem. 270(41):23937-23940 de Kreij A, Venema G, van den Burg B. Substrate specificity in the highly heterogeneous M4 peptidase family is determined by a small subset of amino acids. (2000) J Biol Chem. 275(40):31115-31120 Du RJ, Ho B. Surface localized Heat Shock Protein 20 (HslV) of Helicobacter pylori. (2003) Helicobacter 8(4):257-267 Eaton KA, Brooks CL, Morgan DR, Krakowka S. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. (1991) Infect Immun. 59(7):2470-2475 Eaton KA, Suerbaum S, Josenhans C, Krakowka S. Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. (1996) Infect Immun. 64(7):2445-2448 Fiocca R, Luinetti O, Villani L, Chiaravalli AM, Capella C, Solcia E. Epithelial cytotoxicity, immune responses, and inflammatory components of Helicobacter pylori gastritis. (1994) Scand J Gastroenterol Suppl. 205:11-21 Gong M, Ho B. Prominent role of gamma-glutamyl-transpeptidase on the growth of Helicobacter pylori. (2004) World J Gastroenterol. 10(20):2994-2996 Haas G, Karaali G, Ebermayer K, Metzger WG, Lamer S, Zimny-Arndt U, Diescher S, Goebel UB, Vogt K, Roznowski AB, Wiedenmann BJ, Meyer TF, Aebischer T, Jungblut PR. Immunoproteomics of Helicobacter pylori infection and relation to gastric disease. (2002) Proteomics 2 : 313-324 Hase CC, Finkelstein RA. Bacterial extracellular zinc-containing metalloproteases. (1993) Microbiol Rev. 57(4):823-837 Hickey RM, Twomey DP, Ross RP, Hill C. Production of enterolysin A by a raw milk enterococcal isolate exhibiting multiple virulence factors (2003) Microbiology 149(Pt 3):655-664 Holt C, Sawyer L. Primary and predicted secondary structures of the caseins in relation to their biological functions. (1988) Protein Eng. 2(4):251-9 Hooper NM. Families of zinc metalloproteases. (1994) FEBS Lett. 354(1):1-6 Ichimura T, Yamazoe M, Maeda M, Wada C, Hiraga S. Proteolytic activity of YibP protein in Escherichia coli. (2002) J Bacteriol.184:2595-2602 Ichinose Y, Ehara M, Honda T, Miwatani T. The effect on enterotoxicity of protease purified from Vibrio cholerae O1. (1994) FEMS Microbiol Lett. 115(2-3):265-271 Karita M, Etterbeek ML, Forsyth MH, Tummuru MK, Blaser MJ. Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant. (1997) Infect Immun. 65(10):4158-4164 Kavermann H, Burns BP, Angermuller K, Odenbreit S, Fischer W, Melchers K, Haas R. Identification and characterization of Helicobacter pylori genes essential for gastric colonization. (2003) J Exp Med. 197(7):813-822 Kim KI, Park SC, Kang SH, Cheong GW, Chung CH. Selective degradation of unfolded proteins by the self-compartmentalizing HtrA protease, a periplasmic heat shock protein in Escherichia coli. (1999) J Mol Biol. 294 : 1363-1374 Kovach ME, Hughes KJ, Everiss KD, Peterson KM. Identification of a ToxR-activated gene, tagE, that lies within the accessory colonization factor gene cluster of Vibrio cholerae O395. (1994) Gene148(1):91-95 Lai AC, Tran S, Simmonds RS. Functional characterization of domains found within a lytic enzyme produced by Streptococcus equi subsp. Zooepidemicus. (2002) FEMS Microbiol Lett. 215(1):133-138 Leunk RD, Johnson PT, David BC, Kraft WG, Morgan DR. Cytotoxic activity in broth-culture filtrates of Campylobacter pylori. (1988) J Med Microbiol. 26(2):93-99 Lin SN, Ayada K, Zhao Y, Yokota K, Takenaka R, Okada H, Kan R, Hayashi S, Mizuno M, Hirai Y, Fujinami Y, Oguma K. Helicobacter pylori heat-shock protein 60 induces production of the pro-inflammatory cytokine IL8 in monocytic cells. (2005) J Med Microbiol. 54(Pt 3):225-233 Mahdavi J, Sonden B, Hurtig M, Olfat FO, Forsberg L, Roche N, Angstrom J, Larsson T, Teneberg S, Karlsson KA, Altraja S, Wadstrom T, Kersulyte D, Berg DE, Dubois A, Petersson C, Magnusson KE, Norberg T, Lindh F, Lundskog BB, Arnqvist A, Hammarstrom L, Boren T. Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation. (2002) Science 297(5581):573-578 Marguerite C, Paul D, Stephen D, Richard O’K, Felicity E.B.M, Bruce R.W Brendan D. Helicobacter pylori interacts with the human single-domain trefoil protein TFF1. (2004) Proc Natl Acad Sci U S A.101(19):7409-7414 McGowan CC, NechevaMolecula AS. Promoter analysis of Helicobacter pylori genes with enhanced expression at low pH. (2003) Microbiology 48(5):1225-1239 Merrell DS, Gooddrich ML, Otto G, Tompkins LS, Falkow S. pH-regulastric gene expression of the gastric pathogen Helicobacter pylori. (2003) Infect Immun. 71 (6): 3529-3539 Miyoshi S, Shinoda S. Microbial metalloproteases and pathogenesis. (2000) Microbes Infect. 2(1):91-98 Montecucco C, Papini E, de Bernard M, Zoratti M. Molecular and cellular activities of Helicobacter pylori pathogenic factors. (1999) FEBS Lett. 452(1-2):16-21. Moore JT, Uppal A, Maley F, Maley GF. Overcoming inclusion body formation in a high-level expression system. (1993) Protein Express. Purif. 4: 160-163 Moran AP, Shiberu B, Ferris JA, Knirel YA, Senchenkova SN, Perepelov AV, Jansson PE, Goldberg JB. Role of Helicobacter pylori rfaJ genes (HP0159 and HP1416) in lipopolysaccharide synthesis. (2004) FEMS Microbiol Lett. 241(1):57-65 Negro A, Onisto M, Grassato L, Caenazzo C, Garbisa S. Recombinant human TIMP-3 from Escherichia coli: synthesis, refolding, physico-chemical and functional insights. (1997) Protein Eng.10(5):593-599 Odenbreit S, Till M, Hofreuter D, Faller G, Haas R. Genetic and functional characterization of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric tissue.(1999) Mol Microbiol. 31(5):1537-1548 Odintsov SG, Sabala I, Marcyjaniak M, Bochtler M. Latent LytM at 1.3A resolution. (2004) J Mol Biol. 335(3):775-785 Pedersen LL, Radulic M, Doric M, Abu Kwaik Y. HtrA homologue of Legionella pneumophilia : an indispensable element for intracellular infection of mammalian but not protozoan cells. (2001) Infect Immun. 69(4): 2569-2579 Peters JE, Galloway DR. Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity. (1990) J Bacteriol. (5):2236-2240 Peterson KM, Mekalanos JJ. Characterization of the Vibrio cholerae ToxR regulon : indentification of novel genes involved in intestinal colonization. (1988) Infect Immun. 56(11) : 2822-2829 Poquent I, Saint V, Seznec E, Simoes N, Bolotin A, Gruss A. HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing. (2000) Mol Microbiol. 35(5): 1042-1051 Rawlings ND, Tolle DP, Barrett AJ. MEROPS: the peptidase database. (2004) Nucleic Acids Res. 32(Database issue):D160-164 Rowland M. Transmission of Helicobacter pylori: is it all child's play? (2000) Lancet. 355(9201):332-333 Salama NR, Shepherd B, Falkow S. Global transposon mutagenesis and essential gene analysis of Helicobacter pylori. (2004) J Bacteriol. 186(23): 7926-7935 Sidebotham RL, Worku ML, Karim QN, Dhir NK, Baron JH. How Helicobacter pylori urease may affect external pH and influence growth and motility in the mucus environment: evidence from in-vitro studies. (2003) Eur J Gastroenterol Hepatol. 15(4):395-401 Simmonds RS, Simpson WJ, Tagg JR. Cloning and sequence analysis of zooA, a Streptococcus zooepidemicus gene encoding a bacteriocin-like inhibitory substance having a domain structure similar to that of lysostaphin. (1997) Gene 189(2):255-261 Spiess C, Beil A, Ehrmann M. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. (1999) Cell 97: 339-347 Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton GG, Fleischmann RD, Ketchum KA, Klenk HP, Peterson S, Loftus B, Richardson D, Dodson R, Khalak HG, Glodek A, McKenney K, Fitzegerald LM, Lee N. Adams MD, Hickey EK, Berg DE, Gocayne JD, Utterback TR, Peterson JD, Kelley JM, Cotton MD, Weidman JM, Fujii C, Bowman C, Watthey L, Hayes WS, Borodovsky M, Karp PD, Smith HO, Fraser CM, Venter JC. The complete genome sequence of the gastric pathogen Helicobacter pylori. (1997) Nature 388:539-547 Warren JR, Marshall BJ. Unidentified curved bacilli on gastrc epithelium in active chronic gastritis. (1983) Lancet. 1:1273-1275 Windle HJ, Kelleher D. Identification and characterization of a metalloprotease activity from Helicobacter pylori. (1997) Infect Immun. 65(8):3132-3137 Yamamoto T, Hanawa T, Ogata S, Kamiya S. The Yersinia enterocolitica GsrA stress protein, involved in intracellular survival, is induced by macrophage phagocytosis. (1997) Infect Immun. 65(6):2190-2196 Yoshida N, Granger DN, Evans DJ Jr, Evans DG, Graham DY, Anderson DC, Wolf RE, Kvietys PR. Mechanisms involved in Helicobacter pylori-induced inflammation. (1993) Gastroenterology 105(5):1431-1440 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36721 | - |
dc.description.abstract | 幽門螺旋桿菌 (Helicaobacter pylori) 寄生於人類胃中,目前已知與許多胃部疾病相關,但詳細的致病機制與毒性仍需更進一步的釐清和證明。在微生物中,蛋白酶除了在生存上是必備的成分外,為了能夠寄生在宿主體內,微生物將會發展出針對宿主產生破壞與毒性的功能,例如在營養的取得、毒性因子的合成、分解宿主細胞特定蛋白以便控制宿主的反應以及菌體本身蛋白量與質的控制等。幽門螺旋桿菌26695菌株中有33個基因已被預測出具有蛋白酶的功能,其中我們挑選了tagE、htrA這兩個基因進行探討,這是因為之前的研究並未在蛋白酶功能部分多所著墨,此外也發現這兩個基因轉錄出來的mRNA在酸性環境刺激下都有上升的趨勢。我們在幽門螺旋桿菌NTUH-C1菌株選殖這兩個基因,由於htrA基因在幽門螺旋桿菌中已被證明是essential gene,所以我們僅製作tagE突變株來看對幽門螺旋桿菌的影響。初步發現tagE突變株在生長複製上的表現與野生株無太大的差異,但是經由初步的統計結果及穿透式電子顯微鏡的觀察,我們可以看到突變株的菌體長度較野生株短。之後,為了進一步確定其蛋白酶的活性與功能,我們在大腸桿菌中使用不同的載體,經過表現和純化的過程後得到TagE與HtrA重組蛋白。最後,我們以zymogram這個技術確定HtrA蛋白的確具有蛋白酶的活性,能夠分解受質β-casein ﹔而TagE蛋白則沒有活性的表現。 | zh_TW |
dc.description.abstract | Helicobacter pylori colonizes in human stomach and is recognized as a cause of many gastric diseases. However, the mechanism of pathogenesis and related toxic factors need further study and identification. In microbes, proteases play important roles in many parts of their life cycles. In order to colonize the host, microbes often use their protease activity to be virulence factors, including the acquisition of nutrient, biosynthesis of pathogenetic factors, cleavage key host proteins for mediation of the host response, and the protein quality control. There are 33 genes which have putative protease function in Helicobacter pylori 26695. In this study, we tried to characterize the TagE、HtrA protein functions, because their protease activity is not yet clarify and mRNA expression in high level under acidic stress. We cloned the tagE、htrA genes from Helicobacter pylori NTUH-C1. htrA is proved as an essential gene, so only the tagE-knockout strain was constructed. Then, we compared the wild type and tagE-knockout strain, it’s no distint difference in the growth and replication, but under the transmission electron microscopy and statistics results we observed the phenotype of the tagE-knockout strain has decreased elongation. Moreover, to identify the protease activity, we expressed and purified the TagE and HtrA recombinant protein. Finally, zymogram confirmed that the htrA had the protease activity to degrade β-casein, but tagE didn’t. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T08:12:44Z (GMT). No. of bitstreams: 1 ntu-94-R92445102-1.pdf: 2234783 bytes, checksum: a50341f1e5c926e24d08909346b47df3 (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 目錄………………………………………………………………………………......1
中文摘要……………………………………………………………………………..3 英文摘要……………………………………………………………………………..4 第一章 前言…………………………………………………………………………5 第二章 研究目的……………………………………………………………………11 第三章 實驗材料與方法 3.1 實驗材料 3.1.1 幽門螺旋桿菌 (H. pylori) 菌株………………………………………...12 3.1.2 大腸桿菌 (E. coli) 菌株………………………………………………...12 3.1.3 培養基……………………………………………………………………13 3.1.4 載體 (vector)…………………………………………………………….14 3.1.5 引子………………………………………………………………………15 3.1.6 抗體………………………………………………………………………15 3.2 實驗方法 3.2.1 tagE基因的選殖與分析…………………………………………………16 3.2.2 htrA 基因的選殖與分析………………………………………………...18 3.2.3 以轉位作用得到幽門螺旋桿菌tagE突變株…………………………18 3.2.4 NTUH-C1野生株與tagE突變株生長曲線與外在型態的比較………..20 3.2.5 獲得TagE重組蛋白……………………………………………………21 3.2.6 製作針對TagE蛋白的專一性抗體……………………………………24 3.2.7 獲得HtrA重組蛋白……………………………………………………25 3.2.8 tagE基因的蛋白酶活性分析……………………………………………26 3.2.9 htrA基因的蛋白酶活性確立……………………………………………26 第四章 實驗結果 4.1 tagE基因的選殖與分析…………………………………………………...27 4.2 htrA 基因的選殖與分析…………………………………………………..28 4.3 以轉位作用得到幽門螺旋桿菌tagE突變株……………………………29 4.4 NTUH-C1野生株與tagE突變株生長曲線與外在型態的比較………….29 4.5 獲得TagE重組蛋白………………………………………………………30 4.6 製作針對TagE蛋白的專一性抗體………………………………………31 4.7 獲得HtrA重組蛋白………………………………………………………32 4.8 tagE基因的蛋白酶活性分析……………………………………………32 4.9 htrA基因的蛋白酶活性確立……………………………………………33 第五章 結果討論……………………………………………………………………34 第六章 圖表 圖一A tagE基因定序的結果……………………………………………………40 圖一B TagE與peptidase subfamily M23B蛋白catalytic residues的比對…......41 圖二A htrA基因定序的結果……………………………………………………42 圖二B HtrA與peptidase subfamily S1C蛋白catalytic residues的比對……….43 圖三 tagE突變株的建立………………………………………………………..44 圖四 野生株與tagE突變株pH7.2與5.5環境下生長曲線的比較……………45 圖五 野生株與tagE突變株外在型態的比較…………………………………46 圖六 野生株與tagE突變株菌體長度量化的比較……………………………47 圖七 野生株與tagE突變株於穿透式電子顯微鏡下外在型態的比較………48 圖八 TagE重組蛋白表現與條件嘗試…………………………………………49 圖九 TagE重組蛋白的純化……………………………………………………50 圖十 Anti-TagE Ab的測試……………………………………………………...51 圖十一 HtrA重組蛋白表現與條件嘗試………………………………………52 圖十二 HtrA 重組蛋白的純化…………………………………………………53 圖十三 Zymogram—TagE蛋白活性分析……………………………………54 圖十四 Zymogram—HtrA蛋白活性確立……………………………………55 第七章 附圖 附圖一 幽門螺旋桿菌26695菌株的HP1543基因.............................................56 附圖二 幽門螺旋桿菌26695菌株的HP1019基因……………………………57 附圖三 full length TagE重組蛋白表現質體的建構……………………………58 附圖四 HtrA重組蛋白表現質體的建構………………………………………59 第八章 參考文獻……………………………………………………………………60 | |
dc.language.iso | zh-TW | |
dc.title | 幽門螺旋桿菌蛋白酶的選殖與功能分析 | zh_TW |
dc.title | Cloning and functional characterization of two putative proteases in Helicobacter pylori | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 周綠蘋,鄧述諄,賴信志 | |
dc.subject.keyword | 幽門螺旋桿菌,蛋白酶, | zh_TW |
dc.subject.keyword | Helicobacter pylori,protease,zymogram, | en |
dc.relation.page | 68 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-20 | |
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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