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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李佳音 | |
dc.contributor.author | Pei-Syuan Wu | en |
dc.contributor.author | 吳培萱 | zh_TW |
dc.date.accessioned | 2021-07-10T21:34:41Z | - |
dc.date.available | 2021-07-10T21:34:41Z | - |
dc.date.copyright | 2016-11-01 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-20 | |
dc.identifier.citation | 1. 林慈玲 (2013) 腸炎弧菌胞外蛋白酶受Fur調控及RyhB特性分析。碩士論文,國立台灣大學,台北市。
2. 趙芳筠 (2012)攝鐵調控蛋白及鐵離子對於腸炎弧菌胞外蛋白酶調控之研究。碩士論文,國立台灣大學,台北市。 3. Abdel, Fattah, AzzaM. 2013. 'Production and partial characterization of collagenase from marine Nocardiopsis dassonvillei NRC2aza using chitin wastes', Egyptian Pharmaceutical Journal, 12: 109. 4. Andrews, S. C., A. K. Robinson, and F. Rodriguez-Quinones. 2003. 'Bacterial iron homeostasis', FEMS Microbiol Rev, 27: 215-37. 5. Baker-Austin, C., L. Stockley, R. Rangdale, and J. Martinez-Urtaza. 2010. 'Environmental occurrence and clinical impact of Vibrio vulnificus and Vibrio parahaemolyticus: a European perspective', Environ Microbiol Rep, 2: 7-18. 6. Broberg, C. A., T. J. Calder, and K. Orth. 2011. 'Vibrio parahaemolyticus cell biology and pathogenicity determiants', Microbes Infect, 13: 992-1001. 7. Ceccarelli, D., N. A. Hasan, A. Huq, and R. R. Colwell. 2013. 'Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors', Front Cell Infect Microbiol, 3: 97. 8. Craig, S. A., C. D. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76662 | - |
dc.description.abstract | 腸炎弧菌的胞外蛋白酶為造成人類腹瀉或腸胃炎之潛在致病因子,本次研究觀察胞外蛋白酶基因prtV 上游基因對它的調控,PrtV除了是金屬蛋白酶 (Metalloprotease) 也是一種膠原蛋白酶 (Collagenase),目前發現大部分會產生膠原蛋白酶的細菌皆為致病菌。故本次研究以腸炎弧菌 (Vibrio parahaemolyticus no.93) 為研究菌株,探討腸炎弧菌的金屬蛋白酶PrtV (VPA0459) 基因表現的的調控,本次探討的調控因子有兩項,其中一項為Fur (Ferric uptake regulator; VP0833) 蛋白質對prtV基因轉錄的影響,另一項則為prtV上游基因 (Hypothetical protein, VPA0458) 是否參與prtV基因表現的調控。首先利用即時定量聚合連鎖反應 (qRT-PCR) 檢測發現調控因子Fur和VPA0458 對prtV基因具有負調控情形。再利用報導基因pLuxAB螢光表現分析,同樣發現在細胞內fur和vpa0458 因子對prtV上游啟動子有負調控結果。在其他的文獻顯示弧菌中Fur調控會受到Fe2+濃度影響,因此也探討不同二價鐵離子濃度的情況下Fur的調控情形,以qRT-PCR分析結果顯示在低鐵環境下相較於高鐵生長條件下prtV基因表現下降,因此推測Fur在高鐵環境下會抑制prtV基因表現。prtV上游基因序列有兩段高保守19-bp 序列可能為Fur box binding site,分別命名為Fur box 1以及Fur box 2,電泳延滯(EMSA)和足跡實驗 (Footprinting)實驗證實Fur蛋白質直接結合在prtV啟動子之Fur box 1。 | zh_TW |
dc.description.abstract | The extracellular proteases of Vibrio parahaemolyticus is a potential virulence factor that causes diarrhea or gastroenteritis. In this studies, we discussed the upstream regulation of extracellular proteases prtV gene. PrtV protein is not only a metalloprotease but also collagenase. A variety of bacteria have been known as collagenase producers. Majority of them are pathogenic strains, applying the enzyme as the virulence factor. In this study, we used Vibrio parahaemolyticus no.93 strain to investigate gene expression of PrtV (VPA0459). We were interested in prtV gene expression of two regulators between Fur (Ferric uptake regulator; VP0833) and VPA0458 (hypothetical protein). We designed and performed a series of in vivo and in vitro tests on prtV gene expression. In vivo test, Fur and VPA0458 have negative regulation in qRT-PCR analysis. In vitro test, Fur and VPA0458 have negative regulation in LuxAB activity analysis. In other literature shows that importance of Fe2+ in Vibrio. Therefore, we describe how changed in iron levels influence gene expression through Fur-mediated regulation of prtV. Our result reveals that prtV gene expression was higher in iron-limited cultures than in iron-replete cultures in qRT-PCR analysis. We hypothesized that Fur repressed prtV under iron-replete condition. There are two putative Fur box binding sites containing highly conserved 19-bp sequence on the upstream of prtV gene. They are named Fur box 1 and Fur box 2. We also found that prtV gene expression is subject to negative regulation through the binding of Fur on the Fur box 1 with electrophoretic mobility shift assay and Footprinting assay. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T21:34:41Z (GMT). No. of bitstreams: 1 ntu-105-R03623025-1.pdf: 2993105 bytes, checksum: 5f181d3816f7a20909bd38941d53ad75 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 中文摘要 i
英文摘要 ii 目錄 iv 圖次 viii 附錄表次 x 縮寫表 xi 壹、前言 1 一、腸炎弧菌(Vibrio parahaemolyticus ) 1 二、腸炎弧菌致病情形 2 三、腸炎弧菌的致病因子 3 (1) 熱穩定溶血素 ( Thermostable direct hemolysin, TDH ) 3 (2) 胞外蛋白酶 (Extracellular protease) 5 (3) 膠原蛋白酶 (Collagenase) 6 (5) 腸炎弧菌胞外膠原蛋白酶 7 (6) 霍亂弧菌胞外金屬蛋白酶PrtV 8 四、腸炎弧菌的蛋白質分泌系統 9 五、攝鐵機制 10 (1) 鐵離子 10 (2) 攝鐵調控蛋白 (Ferric uptake regulator, Fur) 11 (3) 弧菌屬之攝鐵調控蛋白研究 12 六、研究動機與目的 13 貳、 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、溶液與緩衝溶液 15 五、實驗使用套組 19 六、儀器設備 20 參、 實驗方法 22 一、DNA技術 22 二、RNA技術 26 三、蛋白質技術 28 四、電泳遷移率實驗 (Electrophoretic mobility shift assay, EMSA) 31 五、建構腸炎弧菌突變株 33 六、南方墨點法 37 七、DNase I 足印法(DNase I footprinting assay) 39 九、生物資訊分析 41 十、統計分析 42 肆、實驗結果 43 一、利用生物資訊學分析vpa0458 親緣性菌株 43 二、利用生物資訊學分析vpa0458 與 prtV (vpa0459) 之間的基因序列 43 三、以qRT-PCR分析prtV、vpa0458、 fur 基因之表現時間點 44 四、建構ΔVPA0458 和ΔVPA0458Δfur 腸炎弧菌突變株 45 五、南方墨點法證明ΔVPA0458 和ΔVPA0458Δfur 腸炎弧菌突變株 46 六、腸炎弧菌野生珠與突變株生長曲線測定結果 47 七、以pLuxAB螢光活性分析確認prtV與vpa0458基因的啟動子 48 八、以qRT-PCR分析腸炎弧菌在不同鐵離子情況下prtV基因表現情形 49 九、以LuxAB螢光分析腸炎弧菌prtV啟動子在VP93野生株與Δfur突變株受調控之影響 50 十、利用電泳遷移率實驗觀察Fur與預測之Fur box 1 和Fur box 2 結合 情形 50 十一、以qRT-PCR 測定在VP93野生株和ΔVPA0458和ΔVPA0458 Δfur和Δfur突變菌株中 prtV基因表現差異 52 十二、以LuxAB螢光分析腸炎弧菌在VP93野生株與ΔVPA0458突變株中VPA0458 調控因子對prtV影響 52 伍、討論 54 陸、結論 56 柒、未來展望 58 捌、參考文獻 59 | |
dc.language.iso | zh-TW | |
dc.title | 腸炎弧菌 prtV基因表現受 Fur及 VPA0458的調控 | zh_TW |
dc.title | Regulation of Vibrio parahaemolyticus prtV gene expression by Fur and VPA0458 | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 邵國銓,廖仁寶 | |
dc.subject.keyword | 腸炎弧菌,膠原蛋白?,調控子, | zh_TW |
dc.subject.keyword | Vibrio parahaemolyticus,collagenase,regulator, | en |
dc.relation.page | 110 | |
dc.identifier.doi | 10.6342/NTU201603262 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-20 | |
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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