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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 | Fang-Yun Chao | en |
dc.contributor.author | 趙芳筠 | zh_TW |
dc.date.accessioned | 2021-06-16T23:06:19Z | - |
dc.date.available | 2017-08-27 | |
dc.date.copyright | 2012-08-27 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-06 | |
dc.identifier.citation | 詹于慧 (2011) 腸炎弧菌胞外蛋白酶 PrtA 調控機制之研究. 碩士論文, 國立臺灣大學, 台北市.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64897 | - |
dc.description.abstract | 腸炎弧菌(Vibrio parahaemolyticus)胞外蛋白酶為潛在之致病因子,其致病能力可能源自於菌體攝取外界養分而造成,其中,攝鐵能力即在細胞存活及生長過程扮演重要且必須之角色。腸炎弧菌膠原蛋白酶 PrtV(VPA0459)基因的上游區域有兩處可能之攝鐵調控蛋白結合位(Fur box),絲胺酸蛋白酶 PrtA(VPA0227)及小 RNA RyhB 上游具一處可能之結合位,故本研究利用一株腸炎弧菌缺乏 tdh 及 trh 之台灣臨床分離菌株, V. parahaemolyticus NO.93 為研究材料,分別建構 fur 突變株及 ryhB過量表現菌株,擬探討胞外蛋白酶 PrtV 及 PrtA 此兩蛋白酶基因之調控是否與鐵離子、攝鐵調控蛋白(Fur),以及 RyhB密切相關。由 FURTA 實驗結果可得知, RyhB 基因上游啟動子區域具有與 Fur 結合之能力。 Q-PCR 結果顯示, Fur 負向調控 prtV 及 prtA 轉錄,但由 FURTA 結果可推測此調控並非由於 Fur 直接結合於此兩蛋白酶上游啟動子區而抑制;而 RyhB 則正向調控 prtV 及 prtA 轉錄。由西方墨點法結果也顯示, PrtV 及 PrtA 皆能夠受 Fur 及鐵離子負向調控,於 RyhB 過量表現菌株也可偵測到較野生株高量之 PrtA 表現,因此推測 Fur 調控 PrtA 表現是經由 RyhB;而 PrtV 雖然能夠受 Fur 及鐵離子的負向調控,但於 RyhB 過量表現菌株中無論在胞內或胞外並無法偵測到較野生株高量之 PrtV 表現,顯示 RyhB 對 PrtV 的調控並不影響其分泌途徑。本篇研究證實腸炎弧菌胞外蛋白酶 PrtV 及 PrtA 會受到 Fur-Fe2+ 及 RyhB所調控,此發現有助於未來探討此系統對於腸炎弧菌致病機制之研究。 | zh_TW |
dc.description.abstract | Vibrio parahaemolyticus causes acute gastroenteritis in humans and it is one of the most important food-borne pathogens in many countries. The putative Fur (ferric uptake regulator) boxes, each containing 19-bp highly conserved sequence, were found in the upstream of promoter regions of prtV encoding metalloprotease (VPA0459), prtA encoding alkaline serine protease (VPA0227) and small RNA RyhB (between VP0105 and VP0106) genes. To clarify whether the production of PrtV and PrtA are regulated by iron, Fur and RyhB, the fur deletion mutant and ryhB overexpression derivatives were generated. The results of FURTA indicated that Fur can bind to a putative Fur box sequence on ryhB and it is functional as recognized by E. coli Fur protein. The Q-PCR showed that the prtV and prtA genes were negatively regulated by Fur protein, but they were positively regulated by small RNA RyhB. Western blot analysis demonstrated that V. parahaemolyticus senses iron availability via Fur to regulate the production of PrtV and PrtA. While the extracellular PrtA was increased in ryhB overexpression strain, the production of PrtV was not elevated neither in the fraction of extracellular nor of intracellular, these results indicated that RyhB did not regulate the secretion of PrtV and PrtA. These observations demonstrate that in V. parahaemolyticus the Fur/RyhB regulatory pathway provides a mechanism to regulate the expression of extracellular protease PrtV and PrtA. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T23:06:19Z (GMT). No. of bitstreams: 1 ntu-101-R99623023-1.pdf: 6012103 bytes, checksum: b737fef081397df2554f982d4f132c21 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 口試委員會審定書……………………………………………………………i
誌謝 ………………………………………………………………………...ii 中文摘要. iii 英文摘要. iv 目錄 …………………………………………………………………………v 表次 ………………………………………………………………………...ix 圖次 …………………………………………………………………………x 附錄圖次 …………………………………………………………………….xii 縮寫表 ……………………………………………………………………xiii 壹、 前言 1 一、 腸炎弧菌(Vibrio parahaemolyticus) 1 1. 概述 1 2. 性狀 2 3. 毒力因子(virulence factors) 2 二、 胞外蛋白酶(extracellular protease) 3 1. 概述 3 2. 腸炎弧菌胞外蛋白酶相關研究 4 3. 膠原蛋白酶 4 4. 腸炎弧菌胞外膠原蛋白酶 PrtV 5 5. 腸炎弧菌絲胺酸蛋白酶 PrtA 6 三、 攝鐵機制 6 1. 概述 6 2. 攝鐵調控蛋白(ferric uptake regulator, Fur) 7 3. 攝鐵調控蛋白所調控的目標基因 7 4. 腸炎弧菌(Vibrio parahaemolyticus)的攝鐵調控蛋白 9 5. 溶藻弧菌(Vibrio alginolyticus)的攝鐵調控蛋白 9 6. 創傷弧菌(Vibrio vulnificus)的攝鐵調控蛋白 10 7. 擬態弧菌(Vibrio mimicus)的攝鐵調控蛋白 10 8. 霍亂弧菌(Vibrio cholera)的攝鐵調控蛋白 11 四、 Small RNA RyhB 12 1. 概述 12 2. RyhB 調控機制 13 3. RyhB 於弧菌屬之研究 14 五、 研究目的與動機 15 貳、 實驗材料與方法 16 I. 實驗材料 16 一、 實驗菌株、質體與引子 16 二、 培養基 16 三、 藥品與試劑 17 四、 各種溶液及緩衝溶液 17 五、 儀器設備 19 II. 實驗方法 21 一、 DNA 技術 21 二、 RNA 技術 25 三、 蛋白質技術 28 四、 生物資訊庫軟體分析 29 五、 建構 Dfur 及 DprtV 突變株 30 六、 建構補償株 34 七、 腸炎弧菌基本生理特性分析 35 八、 攝鐵調控蛋白滴定分析(Fur titration assay, FURTA) 36 參、 實驗結果 38 一、 腸炎弧菌 NO. 93 膠原蛋白酶 PrtV 與相似蛋白之比較 38 二、 PrtV 上游基因及其保守性區域 38 三、 Small RNA ryhB 編碼區及其上游序列 39 四、 Web Logo 軟體分析弧菌屬中已知 Fur box 之保守性及其與 prtV、 prtA 及 ryhB 上游假定 Fur box 比較 40 五、 prtV、 prtA 及 ryhB 上游假定 Fur box 區域及其攝鐵調控蛋白滴定分析(FURTA) 41 六、 建構腸炎弧菌 NO. 93 之 fur 及 prtV 突變株 42 七、 建構腸炎弧菌 NO. 93 之 fur、 prtV 補償株及 WT / p、 WT / pryhB 菌株 43 八、 比較各菌株生長速率與胞外蛋白酶活性表現 44 九、 鐵離子對於胞外蛋白酶活性之影響 44 十、 以 Q-PCR 比較各菌株間 prtV、 prtA 基因轉錄之差異 45 十一、 以西方墨點法觀察 12 小時內腸炎弧菌 NO. 93 中 PrtV 及 PrtA 表現之差異 46 十二、 以西方墨點法觀察 Fur 及鐵離子對於 PrtV 及 PrtA 胞外表現之影響 46 十三、 以西方墨點法觀察各菌株間 PrtV 及 PrtA 胞外表現之差異 47 十四、 以西方墨點法觀察 RyhB 對於 PrtV 及 PrtA 胞外表現之影響 …………………………………………………………………….48 十五、 以西方墨點法觀察 RyhB 對於 PrtV 及 PrtA 胞內表現之影響 …………………………………………………………………….48 肆、 討論 50 一、 FUR/RyhB 調控機制與致病之關係 50 二、 LuxO-OpaR 系統對胞外蛋白酶 PrtV 及 PrtA 之調控 51 三、 腸炎弧菌胞外蛋白酶及其表現之消長情形 51 伍、 結論 53 陸、 參考文獻 54 | |
dc.language.iso | zh-TW | |
dc.title | 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 | zh_TW |
dc.title | Regulation of extracellular protease in Vibrio parahaemolyticus by Iron and Fur | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 賴信志,彭慧玲 | |
dc.subject.keyword | 腸炎弧菌,胞外蛋白酶,攝鐵調控蛋白,RyhB, | zh_TW |
dc.subject.keyword | Vibrio parahaemolyticus,extracellular protease,ferric uptake regulator,Fur,RyhB, | en |
dc.relation.page | 99 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-06 | |
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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