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  1. NTU Theses and Dissertations Repository
  2. 生物資源暨農學院
  3. 動物科學技術學系
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42302
完整後設資料紀錄
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dc.contributor.advisor朱有田
dc.contributor.authorPei-Hsuan Chungen
dc.contributor.author鍾佩軒zh_TW
dc.date.accessioned2021-06-15T00:58:42Z-
dc.date.available2018-07-08
dc.date.copyright2008-08-08
dc.date.issued2008
dc.date.submitted2008-08-01
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張德銘、黃勤鎮、林鉅鋃。2000。『抗生素濫用影響國人健康』專案調查報告。凱倫出版社,台北市。
潘明正、蔡向榮。1982。簡明家畜傳染病學。藝軒圖書出版社,台北市。
劉榮標。1993。獸醫微生物學(上冊)。藝軒圖書出版社,台北市。
歐柏榮。2003。基因轉殖技術在農業上之應用。教育部顧問室出版,台北市。
Barber, M. and M. Rozwadowska-Dowzenko. 1948. Infection by penicillin-resistant Staphylococci. Lancet ii: 641-644.
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Bramley, A. J. and F. H. Dodd. 1984. Reviews of the progress of dairy science: Mastitis control-progress and prospects. J. Dairy Res. 51:481-512.
Bramley, A. J. and R. Foster. 1990. Effects of lysostaphin on Staphylococcus aureus infections of the mouse mammary gland. Res. Vet. Sci. 49: 120-121.
Climo, M. W., L. Patron, B. P. Goldstein, and G. L. Archer. 1998. Lysostaphin treatment of experimental methicillin-resistant Staphylococcus aureus aortic valve endocarditis. Antimicrob. Agents Chemother. 42: 1355-1360.
Contreras, A., D. Sierra, J. C. Corrales, A. Sanchez, and J. Macro. 1996. Physiological threshold of somatic cell count and California Mastitis Test for diagnosis of caprine subclinical mastitis. Small Rumin. Res. 21: 259-264.
Cooper, G. M. and R.E. Hausman. 2007. The Cell: a Molecular Approach, 4th ed. ASM Press. Washington, D. C.
Craven, N. and J. C. Anderson. 1984. Phagocytosis of Staphylococcus aureus by bovine mammary gland macrophages and intracellular protection from antibiotic action in vitro and in vivo. J. Dairy Res. 51: 513-523.
Dajcs, J. J., B. A. Thibodeaux, D. O. Girgis, M. D. Shaffer, S. M. Delvisco, and R. J. O’Callaghan. 2002. Immunity to lysostaphin and its therapeutic value for ocular MRSA infections in the rabbit. Invest. Ophthalmol. Vis. Sci. 43: 3712-3716.
Daley, M. J. and E. R. Oldham. 1992. Lysostaphin: immunogenicity of locally administered recombinant protein used in mastitis therapy. Vet. Immunol. Immunopathol. 31: 301-312.
Dodd, F. H. and T. K. Griffin. 1975. The role of antibiotic treatment at drying off in the control of mastitis. Int. Dairy Fed. 85: 282-302.
Ehlert, K., W. Schroder, and H. Labischinski. 1997. Specificities of FemA and FemB for different glycine residues: FemB cannot substitute for FemA in Staphylococcal peptidoglycan pentaglycine side chain formation. J. Bacteriol. 179: 7573-7576.
Esslemont, D. and M. Kossaibati. 2002. Mastitis: how to get out of the dark ages. Vet. J. 164: 85-86.
Fan, W., K. Plaut, A. J. Bramley, J. W. Barlow, and D. E. Kerr. 2002. Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland. J. Dairy Sci. 85: 1709-1716.
Gutierrez-Adan, A., E. A. Maga, H. M. Meade, C. F. Shoemaker, J. F. Medrano, G. B. Anderson and J. D. Murray. 1996. Alteration of physical characteristics of milk from bovine kappa-casein transgenic mice. J. Dairy Sci. 79: 791-799.
Heinrich, P., R. Rosenstein, M. Böhmer, P. Sonner, and F. Götz. 1987. The molecular organization of the lysostaphin gene and its sequences repeated in tandem. Mol. Gen. Genet. 209: 563-569.
Hennighausen, L. and G. W. Robinson. 2001. Signaling pathways in mammary gland development. Dev. Cell. 1: 467-475.
Jurgeit, A., C. Berlato, P. Obrist, C. Ploner, P. Massoner, J. Schmölzer, M. C. Haffner, H. Klocker, L. A. Huber, S. Geley, and W. Doppler. 2007. Insulin-like growth factor-binding protein-5 enters vesicular structures but not the nucleus. Traffic 8: 1815-1828.
Kerr, D. E., K. Plaut, A. J. Bramley, C. M. Williamson, A. J. Lax, K. Moore, K. D. Wells, and R. J. Wall. 2001. Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice. Nat. Biotechnol. 19: 66-70.
Kim, S. J., B. H. Sohn, S. Jeong, K. W. Pak, J. S. Park, I. Y. Park, T. H. Lee, Y. H. Choi, C. S. Lee, Y. M. Han, D. Y. Yu, and K. K. Lee. 1999. High-level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence. J. Biochem. 126: 320-325.
Kusuma, C., A. Jadanova, T. Chanturiya, and J. F. Kokai-Kun. 2007. Lysostaphin-resistant variants of Staphylococcus aureus demonstrate reduced fitness in vitro and in vivo. Antimicrob. Agents Chemother. 51: 475-482.
Kusuma, C. M. and J. F. Kokai-Kun. 2005. Comparison of four methods for determining lysostaphin susceptibility of various strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 49: 3256-3263.
Lammers, A., P. J. M. Nuijten, E. Kruijt, N. Stockhofe-Zurwieden, U. Vecht, H. E. Smith, and F. G. van Zijderveld. 1999. Cell tropism of Staphylococcus aureus in bovine mammary gland cell cultures. Vet. Microbiol. 67: 77-89.
Lawrence, T. L. J. and V. R. Fowler. 2002. Growth of Farm Animals. 2nd ed. CAB Intl. Wallingford. England.
Le Provost, F., M. Nocart, G. Guerin, and P. Martin. 1994. Characterization of the goat lactoferrin cDNA: assignment of the relevant locus to bovine U12 synteny group. Biochem. Biophys. Res. Commun. 203: 1324-1332.
Levine, D. P., B. S. Fromm, and B. R. Reddy. 1991. Slow response to vancomycin or vancomycin plus rifampin in methicillin-resistant Staphylococcus aureus endocarditis. Ann. Intern. Med. 115: 674-680.
Levy, S. B. 2002. The Antibiotic Paradox: How the Misuse of Antibiotics Destroys Their Curative Powers, 2nd ed. Perseus publication. Cambridge, MA.
Lewin, B. 2000. Genes VII. Oxford University Press Inc. New York.
Marley, E. F., C. Mohla, and J. M. Campos. 1995. Evaluation of E-test for determination of antimicrobial MICs for Pseudomonas aeruginosa isolates from cystic fibrosis patients. J. Clin. Microbiol. 33: 3191-3193.
Natzke, R. P. and R. W. Everett. 1975. The elimination of mastitis by culling. Int. Dairy Fed. 85: 303-310.
Nottle, M. B., H. Nagashima, P. J. Verma, Z. T. Du, C. G. Grupen, S. M. McIlfatrick, R. J. Ashman, M. P. Harding, C. Giannakis, P. L. Wigley, I. G. Lyons, D. T. Harrison, B. G. Luxford, R. G. Campbell, R. J. Crawford, and A. J. Robins. 1998. Production and analysis of transgenic pigs containing a metallothionein procine growth hormone gene construct. In J. D. Murray, G. B. Anderson, A. M. Oberbauer, M. M. McGloughlin (eds). Transgenic Animals in Agriculture. CAB Intl. Wallingford. England. 145-156.
Oldham, E. R. and M. J. Daley. 1991. Lysostaphin: use of a recombinant bactericidal enzyme as a mastitis therapeutic. J. Dairy Sci. 74: 4175-4182.
Palmiter, R. D., R. L. Brinster, R. E. Hammer, M. E. Trumbauer, M. G. Rosenfeld, N. C. Birnberg, and R. M. Evans. 1982. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth fusion gene. Nature 300: 611-615.
Poutrel, B. and C. Lerondelle. 1983. Cell content of goat milk: California Mastitis Test, Coulter Counter, and Fossomatic for predicting half infection. J. Dairy Sci. 66:2575-2579.
Recsei, P. A., A. D. Gruss, and R. P. Novick. 1987. Cloning, sequence, and expression of the lysostaphin gene from Staphylococcus simulans. Proc. Natl. Acad. Sci. USA 84: 1127-1131.
Rudolph, N. S. 1999. Biopharmaceutical production in transgenic livestock. Trends Biotechnol. 17: 367-374.
Salter, D. W. and L. B. Crittenden. 1989. Artificial insertion of a dominant gene for resistance to avian leucosis virus into the germ line of the chicken. Theor. Appl. Genet. 77: 457-461.
Schindler, C. A. and V. T. Schuhardt. 1964. Lysostaphin: a new bacteriolytic agent for the Staphylococcus. Proc. Natl. Acad. Sci. USA 51: 414-421.
Schuhardt, V. T. and C. A. Schindler. 1964. Lysostaphin therapy in mice infected with Staphylococcus aureus. J. Bacteriol. 88: 815-816.
Small, P. M. and H. F. Chambers. 1990. Vancomycin for Staphylococcus aureus endocarditis in intravenous drug users. Antimicrob. Agents Chemother. 34: 1227-1231.
Sordillo, L. M. and K. L. Streicher. 2002. Mammary gland immunity and mastitis susceptibility. J. Mammary Gland Biol. Neoplasia. 7: 135-146.
Sutra, L. and B. Poutrel. 1994. Virulence factors involved in pathogenesis of bovine intramammary infections due to Staphylococcus aureus. J. Med. Microbiol. 40: 79-89.
van Berkel, P. H., M. M. Welling, M. Geerts, H. A. Van Veen, B. Ravensbergen, M. Salaheddine, E. K. Pauwels, F. Pieper, J. H. Nuijens, and P. H. Nibbering. 2002. Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat. Biotechnol. 20: 484-487.
von Heijne, G. 1988. Transcending the impenetrable: how proteins come to terms with membranes. Biochim. Biophys. Acta. 947: 307-333.
Wall, R. J., A. M. Powell, M. J. Paape, D. E. Kerr, D. D. Bannerman, V. G. Pursel, K. D. Wells, N. Talbot, and H. W. Hawk. 2005. Genetically enhanced cows resist intramammary Staphylococcus aureus infection. Nat. Biotechnol. 23: 445-451.
Wang, Q., Z. Huang, M. J. Chen, S. Z. Huang, and Y. T. Zeng. 2001. GenBank Accession no. AF409096. [Capra hircus beta-casein precursor (csn2) gene, complete cds]. Online. Available: http:// www.ncbi.nlm.nih.gov/.
Williamson, C. M., A. J. Bramley, and A. J. Lax. 1994. Expression of the lysostaphin gene of Staphylococcus simulans in a eukaryotic system. Appl. Environ. Microbiol. 60: 771-776.
Wurm, F. M. 2004. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22: 1393-1398.
Ziomek, C. A. 1998. Commercialization of proteins produced in mammary gland. Theriogenology 49: 139-144.
dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42302-
dc.description.abstractMastitis is an inflammatory reaction of mammary gland usually caused by a microbial infection and is the most prevalent disease of dairy cows and goats. However, Staphylococcus aureus (S. aureus), which currently accounts for 15-30% of mastitis, has been proved more difficult to be controlled using standard management practices. This disease, in addition to causing animal distress, dairy processors also incur losses from detrimental changes in milk composition that accompany mammary gland inflammation. These changes include: decrease of milk production and milk quality. But, the cure rate for treatment of S. aureus mastitis with beta-lactam antibiotics is often less than 15%. Therefore, to develop a novel antibacterial protein is required to cure mastitis infections.
Lysostaphin, a 25 kDa glycylglycine endopeptidase produced by Staphylococcus simulans, can specifically cleave bond between the third and fourth glycines of the pentaglycine cross bridge of the staphylococcal cell wall, and cause the rapid lysis of actively growing staphylococci. It has been demonstrated that lysostaphin treatment significantly reduced viable S. aureus in a mouse and a bovine mastitis model. A lysostaphin transgenic goat was established and showed resistant to mastitis infection, but the detail biological characterization of the recombinant lysostaphin expression in mammary epithelial cells is still unknown.
In order to investigate whether the recombinant lysostaphin could follow the common protein secretory pathway and perform its normal biological function after posttranslational modification in goat mammary epithelial cells, a telomerase-immortalized caprine mammary epithelial cell line (CMEC) was used as a platform to study the biological characteristics of recombinant lysostaphin protein. In an attempt to observe production and secretory pathway of lysostaphin, three plasmids containing recombinant lysostaphins separately fused with beta-casein, lactoferrin and prokaryotic signal peptides were constructed to examine the efficiency of secretion into culture medium. Also, to investigate whether lysostaphin is modified after translation in CMEC, total proteins and mediums were obtained from the CMEC transfected with recombinant plasmids to examine the expression of lysostaphin in CMEC. The beta-casein and lactoferrin signal peptides fused recombinant lysostaphins were detected both in medium and cell extract, indicated both of them could be secreted into medium. The localizations of recombinant lysostaphin proteins in CMEC were examined by immunofluorescence staining after transfections, data revealed the eukaryotic signal peptides directed secretion of the lysostaphin through the endoplasmic reticulum and Golgi apparatus in the CMEC, but prokaryotic signal peptide could not. A large shift in molecular weight were also observed in these recombinant proteins expressing in CMEC, suggesting that lysostaphin might be glycosylated in CMEC before secretion. To determine whether the modification was glycosylation, site-directed mutagenesis was performed to remove N-linked glycosylation sites from the recombinant lysostaphin-expression plasmids. The N-linked glycosylation sites mutated recombinant protein decreased shift in electrophoretic mobility compared to that of unmodified recombinant proteins, indicated the recombinant proteins were glycosylated in CMEC. It seemed that these mutated recombinant proteins secreted in culture medium were proved to possess staphylolytic activity based on bacterial plate assay, but not detected in wild type recombinant proteins, indicating that glycosylation was responsible for the inactivity of lysostaphin in CMEC.
Together these results, suggest that the mutated recombinant proteins may possess normal biological function of antibacterial in caprine mammary epithelial cells. The detailed biological effect on caprine mammary epithelial cells needs further investigation.
en
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Previous issue date: 2008
en
dc.description.tableofcontents口試委員會審定書.........................................I
目錄....................................................II
圖次.....................................................V
表次..................................................VIII
誌謝....................................................IX
摘要....................................................XI
英文摘要..............................................XIII
第一章 前言..............................................1
第二章 文獻探討..........................................3
一、乳腺之發育.......................................3
(一) 乳腺構造與發育..............................3
(二) 山羊乳乳蛋白質之組成分......................7
二、乳房炎之發生.........................................7
(一) 乳房炎之定義................................7
(二) 乳房炎之分類................................8
1. 臨床型乳房炎..............................8
(1) 急性乳房炎............................8
(2) 壞疽性乳房炎..........................8
2. 亞臨床型乳房炎............................9
(三) 乳房炎病原菌及其感染途徑....................9
(四) 治療與預防乳房炎之方法.....................10
1. 維持擠乳設備及其環境之清淨...............10
2. 擠乳後之乳頭清潔.........................10
3. 乳房炎之治療.............................11
4. 淘汰持續受感染之動物.....................11
5. 乳房炎之預防.............................11
三、治療乳房炎之抗生素..............................12
(一) 抗生素介紹.................................12
(二) 抗生素使用之問題...........................12
1. 金黃色葡萄球菌存活於宿主細胞中...........12
2. 葡萄球菌抗藥性菌株之出現.................13
四、溶葡萄球菌酶 (lysostaphin)......................14
(一) Lysostaphin之發現及其基因結構..............14
1. Lysostaphin簡介..........................14
2. Lysostaphin之基因結構....................15
(二) Lysostaphin之重要性........................17
1. Lysostaphin之功能用途....................17
2. 轉殖基因動物產製之lysostaphin............17
3. Lysostaphin之安全性......................18
(三) 抗Lysostaphin菌株之出現....................19
五、基因轉殖動物在農業上之應用......................20
(一) 應用上之優點...............................20
1. 改善牛乳之組成分.........................20
2. 改善家畜之生長性能.......................21
3. 提高經濟動物的抗病力.....................21
4. 生產生物藥劑.............................22
(二) 產製轉基因動物之研究考量...................24
六、乳蛋白訊息胜肽與蛋白質之運輸....................24
七、乳蛋白訊息胜肽與醣基化蛋白質之運輸..............25
第三章 材料與方法.......................................26
第四章 結果.............................................57
第五章 討論.............................................90
第六章 結論.............................................94
第七章 參考文獻.........................................95
附錄...................................................101
dc.language.isozh-TW
dc.title自Staphylococcus simulans選殖溶葡萄球菌酶基因及其
在不朽化山羊乳腺上皮細胞之功能性研究
zh_TW
dc.titleLysostaphin gene cloned from Staphylococcus simulans and its functional study in immortal caprine mammary epithelial cellsen
dc.typeThesis
dc.date.schoolyear96-2
dc.description.degree碩士
dc.contributor.coadvisor姜延年
dc.contributor.oralexamcommittee徐濟泰,陳銘正,吳希天
dc.subject.keywordStaphylococcus simulans,溶葡萄球菌&#37238,不朽化山羊乳腺上皮細胞,zh_TW
dc.subject.keywordStaphylococcus simulans,lysostaphin,immortal caprine mammary epithelial cells,en
dc.relation.page107
dc.rights.note有償授權
dc.date.accepted2008-08-02
dc.contributor.author-college生物資源暨農學院zh_TW
dc.contributor.author-dept動物科學技術學研究所zh_TW
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