朱有田臺灣大學:動物科學技術學研究所鍾佩軒Chung, Pei-HsuanPei-HsuanChung2010-05-112018-06-292010-05-112018-06-292008U0001-3007200810160500http://ntur.lib.ntu.edu.tw//handle/246246/182045Mastitis 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.口試委員會審定書.........................................I錄....................................................II次.....................................................V次..................................................VIII謝....................................................IX要....................................................XI文摘要..............................................XIII一章 前言..............................................1二章 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、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..............25三章 材料與方法.......................................26四章 結果.............................................57五章 討論.............................................90六章 結論.............................................94七章 參考文獻.........................................95錄...................................................101application/pdf11647634 bytesapplication/pdfen-USStaphylococcus simulans溶葡萄球菌酶不朽化山羊乳腺上皮細胞lysostaphinimmortal caprine mammary epithelial cellsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182045/1/ntu-97-R95626005-1.pdf