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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周綠蘋(Lu-Ping Chow) | |
| dc.contributor.author | Bo-Shih Huang | en |
| dc.contributor.author | 黃伯仕 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:50:11Z | - |
| dc.date.available | 2020-03-12 | |
| dc.date.copyright | 2015-03-12 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-01-22 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55179 | - |
| dc.description.abstract | 中文摘要 幽門螺旋桿菌是胃癌主要的致病因子。先前的研究顯示幽門螺旋桿菌GroES促使胃癌上皮細胞細胞增生以及前發炎因子─介白素8之釋放。幽門螺旋桿菌GroES包含118個胺基酸，位於胺基端的1到90個殘基者稱為功能域A，其與其他種細菌的GroES有同源性；而具獨特性位於羧基端的91到118個殘基者稱為功能域B，其對於鎳離子有很高的親和力。 幽門螺旋桿菌GroES曾經被報導具有六個富有保留性之硫胺酸，會形成三對雙硫鍵，分別是: C51/C53, C94/C111, C95/C112，而後二者導致功能域B中特殊的環狀結構。除此之外，每一個幽門螺旋桿菌GroES 功能域B結合兩個鎳離子。 我們發現在幽門螺旋桿菌GroES處理10 mM DTT之後其引發胃上皮細胞KATO-III細胞株中介白素8之釋放有顯著下降。另外，截斷蛋白包括△幽門螺旋桿菌GroES(1-90)、(1-94)、(1-100)、(1-106)都無法導致介白素8之釋放。這解釋了雙硫鍵對於幽門螺旋桿菌GroES導致介白素8之釋放之重要性。我們更進行了幽門螺旋桿菌GroES點突變以建構C111A, C112A 和C111A/C12A突變體。 前面兩者保留了導致介白素8釋放之能力，但後者卻顯著缺失。 紫外光圓二色光譜顯示C111A/C12A其β轉角以及不規則捲曲比幽門螺旋桿菌GroES原種或是C111A, C112A高了一成。 因此我們認為幽門螺旋桿菌GroES的構型導致KATO-III細胞株中介白素8釋放之能力。 我們同時也發現在幽門螺旋桿菌GroES處理5mM EDTA之後其引發KATO-III細胞株中介白素8之釋放有顯著下降， 可見光圓二色光譜也顯示幽門螺旋桿菌GroES處理不同濃度的EDTA之後， 其鎳離子結合能力呈現與劑量相關下降的趨勢。另外，截斷蛋白△幽門螺旋桿菌GroES(1-112)也失去了引發介白素8之釋放。 這顯示三個組胺酸H113,、H115和H118可能在幽門螺旋桿菌GroES之鎳離子結合上是非常重要的。 我們進一步建構了三個組胺酸變異體: H113、H115和H118A，它們引發介白素8之釋放的能力分別是原種的21 %、81% 和 35%。 可見光圓二色光譜顯示其鎳離子結合能力則分別是原種的0 %、85% 和 40%。 為了找出負責鎳離子配位鍵結的其他組胺酸，我們建構了另外三個組胺酸: H100、H102和H104A，它們引發介白素8之釋放的能力分別是原種的40 %、50% 和 100%。 可見光圓二色光譜顯示其鎳離子結合能力則是原種的70%、100% 和 100%。 我們認為幽門螺旋桿菌GroES的原種、H104A、和H115A的構型導致其鎳離子結合能力，因此使其能夠引發KATO-III細胞株中介白素8之釋放。相反的，H100A、H113A和H118A之構型改變導致其鎳離子缺失，因此失去其引發介白素8之釋放的能力。 然而H102A其引發介白素8之釋放能力則與鎳離子結合無關。 最後，我們發現雙硫鍵和鎳離子兩者對於幽門螺旋桿菌GroES之構型及引發KATO-III細胞株中介白素8釋放之能力是缺一不可的。 | zh_TW |
| dc.description.abstract | Abstract Helicobacter pylori is the main causative agent of gastric cancer. Previous study showed that H. pylori GroES induced proinflammatory cytokine IL-8 in gastric epithelial cells. H. pylori GroES consists of 118 amino acids divided into two domains: an N-terminal domain (domain A, residues1-90), which is homologous with other bacterial GroES, and a unique C-terminal domain (domain B, residue 91-118), which exhibits a high affinity for nickel. It was found that H. pylori GroES has six cysteins, forming three disulfide bonds including C51/C53, C94/C111, C95/C112 and the last two result in unique closed loop structure for the domain B. Additionally, each H. pylori GroES domain B binds two nickel ions. We found that the IL-8 secretion is significantly reduced in gastric epithelial cells KATO-III cells after H. pylori GroES treated with 10 mM DTT. Moreover, the truncacted H. pylori GroES mutants including △H. Pylori GroES(1-90), △H. Pylori GroES(1-94), △H. Pylori GroES(1-100) and △H. Pylori GroES(1-106) are unable to iniduce IL-8 secretion as well. It suggests the crucial role of disulfide bonds on H. pylori GroES ability to cause IL-8 secretion. We further performed the site directed mutagenesis for H. pylori GroES to construct C111A, C112A and C111A/C12A mutants. The former two remain their abilty to cause IL-8 secretion, but the latter significantly loses it. From the far UV-CD spectrum of these mutants, the ratio of β turn and the random coil in the C111A/C12A is 10 % higher than the H. pylori GroES C111A, C112A and wild type. Therefore, we concluded that the H. pylori GroES conformation attributes to its ability to cause IL-8 secretion in KATO-III cells. We also found that the IL-8 secretion is reduced significantly in KATO-III cells after H. pylori GroES treated with 5 mM EDTA. The vis-CD spectrum shows the ability to bind nickel ions are in a dose-dependent manner decrease when H. pylori GroES treated with different concentration of EDTA. Moreover, the truncacted H. pylori GroES mutant △H. Pylori GroES(1-112) loses its ability to cause IL-8 secretion as well. It shows that the three histidines H113, H115 and H118 might be crucial for nickel ions binding to H. pylori GroES. We further constructed three H. pylori GroES mutants including H113A, H115 and H118A. The ability to cause IL-8 release is 21 % , 81% and 35% comparing to wild type, respectively. The vis-CD spectrum shows their ability to bind nickel ions are 0%, 85% and 40% comparing to wild type. In order to find out other histidines responsible for the nickel coordination, we constructed three other mutants including H100A, H102 A, H104A. The ability to cause IL-8 release is 40 % , 50% and 100% comparing to wild type, respectively. The vis-CD spectrum shows their ability to bind nickel ions are 70%, 100% and 100% comparing to wild type. We found that the conformation of H. pylori GroES wild type, H104A, H115A result in nickel binding ability and thus lead to its ability to cause IL-8 secretion in KATO-III cells. In contrast, conformational change of H100A, H113A and H118A result in nickel dissociation and thus lose their ability to cause IL-8 secretion. However, the ability to cause IL-8 secretion of H102A is independent of its nickel binding ability. In conclusion, we found that both of the disulfide bonds and the nickel ions are indispensible to H. pylori GroES conformation and thus ability to cause IL-8 secretion in KATO-III cells. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T03:50:11Z (GMT). No. of bitstreams: 1 ntu-104-R01442014-1.pdf: 2739776 bytes, checksum: 49093dd1d7d36b4520d1759686cc3ce8 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 目錄 口試委員審定書................................................................................................... IV 中文摘要 V Abstract VII 縮寫表.....................................................................................................................IX 第一章 導論 1 第一節 幽門螺旋桿菌 1 1.1 幽門螺旋菌之型態與特徵 1 1.2 幽門螺旋桿菌之流行病學 1 1.3 幽門螺旋桿菌所造成宿主之免疫反應 2 1.4 幽門螺旋桿菌所造成之疾病 3 1.5 幽門螺旋桿菌之致病因子 5 第二節 H. pylori GroES之結構 11 第三節 圓二色光譜術之應用 12 3.1 蛋白二級結構分析 14 3.2 金屬結合分析 14 第四節 研究動機、目的與策略 15 第二章 實驗材料 17 第一節 大腸桿菌及質體 17 第二節 藥品 17 第三節 試劑組 19 第四節 儀器裝置 20 第三章 實驗方法 21 第一節 點突變 21 1.1 引子設計 21 1.2 聚合酶連鎖反應 22 1.3 洋菜膠體電泳 23 1.4 PCR產物之接合反應 24 1.5 製備勝任細胞 24 1.6 轉型作用……………………………………………………………………25 1.7 DNA定序 25 第二節 重組蛋白之表現與純化 26 2.1 重組蛋白之表現 26 2.2 重組蛋白之純化 26 2.3 膠體過濾層析 27 2.4 重組蛋白定量 28 2.5 Sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) 28 第三節 功能性測試 30 3.1 重組蛋白去內毒素 30 3.2 KATO-III細胞培養 31 3.3 ELISA 32 第四節 圓二色光譜術 32 4.1 儀器及分析軟體 32 4.2 樣品置備 34 第四章 結果 35 第一節 探討雙硫鍵對於H. Pylori GroES引發發炎反應之能力扮演之角色35 1.1 重組蛋白His-tagged H. pylori. GroES 表現及純化……………………35 1.2 處理DTT之H. Pylori GroES功能性測試 35 1.3 H. pylori GroES截斷蛋白(truncated protein)之功能性測試 36 1.4 處理DTT之H. Pylori GroES遠紫外光圓二色光譜 36 1.5 H. pylori GroES變異體C111A, C112A and C111A/C112A純化 37 1.6 C111A, C112A和C111A/C112A之功能性測試 37 1.7 C111A, C112A和C111A/C112A遠紫外光圓二色光譜 37 第二節 探討鎳離子對於H. Pylori GroES引發發炎反應之能力扮演之角色38 2.1 處理EDTA之H. Pylori GroES功能性測試 38 2.2 處理EDTA之H. Pylori GroES可見光圓二色光譜 38 2.3 截斷蛋白△H. Pylori GroES (1-112)之功能性測試 39 2.4 H. pylori GroES變異體H113A、 H115A和H118A之純化 39 2.5 H113A、H115A和H118A之功能性測試 39 2.6 H113A、H115A和H118A可見光圓二色光譜 39 2.7 H113A、H115A和H118A遠紫外光圓二色光譜 40 2.8 H113A、H115A和H118A非還原性膠體電泳 41 2.9 H. pylori GroES變異體H100A、 H102A和H104A之建構與純化 41 2.10 H100A、H102A和H104A之功能性測試 42 2.11 H100A、H102A和H104A可見光圓二色光譜 42 2.12 H100A、H102A和H104A遠紫外光圓二色光譜 42 2.13 H100A、H102A和H104A非還原性膠體電泳 43 第五章 討論 44 第一節 圓二色光譜術技術探討 44 第二節 H. pylori GroES與其硫胺酸變異體之二級結構組成和引發發炎能力 之關係探討 45 第三節 H. pylori GroES與其組胺酸變異體之鎳離子結合、二級結構組成和引發發炎能力之關係探討 45 第四節 H. pylori GroES功能域B之鎳離子結合模型 47 第五節 總結與未來展望 51 第六章 參考文獻 53 圖表 63 附錄 77 | |
| dc.language.iso | zh-TW | |
| dc.subject | 介白素8 | zh_TW |
| dc.subject | 多組胺酸序列 | zh_TW |
| dc.subject | 鎳離子配位鍵結 | zh_TW |
| dc.subject | 可見光圓二色光譜 | zh_TW |
| dc.subject | 羧基端 | zh_TW |
| dc.subject | 幽門螺旋桿菌 | zh_TW |
| dc.subject | 致病因子 | zh_TW |
| dc.subject | H. pylori GroES | zh_TW |
| dc.subject | 發炎反應 | zh_TW |
| dc.subject | 雙硫鍵 | zh_TW |
| dc.subject | H. pylori GroES | en |
| dc.subject | inflammatory response | en |
| dc.subject | carboxyl terminus | en |
| dc.subject | disulfide bond | en |
| dc.subject | interleukin 8 | en |
| dc.subject | polyhistidine sequence | en |
| dc.subject | nickel coordination | en |
| dc.subject | visible circular dichroism | en |
| dc.subject | Helicobacter Pylori | en |
| dc.subject | virulence factor | en |
| dc.title | 探討幽門螺旋桿菌GroES之金屬離子及雙硫鍵其引發發炎反應扮演之角色 | zh_TW |
| dc.title | Understanding the role of metal ion and disulfide bond in Helicobacter pylori GroES-induced inflammatory response | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 曾秀如(Shiou-Ru Tzeng) | |
| dc.contributor.oralexamcommittee | 徐駿森(Chun-Hua Hsu),詹迺立(Nei-Li Chan) | |
| dc.subject.keyword | 幽門螺旋桿菌,H. pylori GroES,致病因子,發炎反應,羧基端,雙硫鍵,介白素8,多組胺酸序列,鎳離子配位鍵結,可見光圓二色光譜, | zh_TW |
| dc.subject.keyword | Helicobacter Pylori,H. pylori GroES,virulence factor,inflammatory response,carboxyl terminus,disulfide bond,interleukin 8,polyhistidine sequence,nickel coordination,visible circular dichroism, | en |
| dc.relation.page | 87 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-01-22 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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