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研究生:陳郁伶
研究生(外文):Yu-Ling Chen
論文名稱:腸出血性大腸桿菌O157:H7之致病島嶼L0038功能性研究以及利用螢光壽命顯微技術研究第三型分泌系統蛋白質之交互作用
論文名稱(外文):Characterization of L0038 Encoded in the Pathogenicity Island of Enterohemorrhagic Escherichia coli O157:H7 and Researching of Protein-Protein Interaction in Type III Secretion System by FLIM/FRET
指導教授:許萬枝許萬枝引用關係
指導教授(外文):Wan-Jr Syu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:68
中文關鍵詞:腸出血性大腸桿菌
外文關鍵詞:L0038
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出血性大腸桿菌O157:H7簡稱EHEC,屬於致病性大腸桿菌之一,感染人體後會造成出血性腹瀉以及更嚴重的溶血性尿毒症候群。EHEC利用第三型分泌系統將作用蛋白送入腸道上皮細胞內,細胞因此產生平台狀隆起,使細菌能更緊密貼附於腸道細胞表面。位於EHEC染色體上的致病島嶼LEE含有41個開放性閱讀框架,其中部份的開放性閱讀框架之功能已被定義但仍有些功能還待研究。l0038是致病島嶼上一段功能未知的開放閱讀框,其可合成152個胺基酸,等電點約為5.85的蛋白質。在本論文研究中發現,在染色體刪除l0038基因的剔除突變株中EspA表現量下降並且失去分泌能力,因而推測L0038可能是組成第三型分泌系統的結構蛋白質之一。然而此突變株在利用質體回補表現L0038卻無法回復野生株的分泌表現型,前述推測尚待更多實驗證實。此外,在細菌雙雜合系統當中,我們發現L0038與EscU以及EscU與L0050兩兩之間具有交互作用,由於L0050已被確認為維持EspA穩定性的重要因子之一,因此推測這三個蛋白質之間的連結與EspA的穩定性可能相關。另一方面,第三型分泌系統參與蛋白質之間的交互作用影響其作用與功能甚巨,然而目前仍無適當技術可分析細菌活體中的蛋白質交互作用。我們嘗試將FRET/FLIM技術運用在EHEC第三型分泌系統中蛋白質交互作用之分析。本實驗藉由已被證實互相結合的CesT與Tir做為分析對象,分別將之融合紅色以及綠色螢光蛋白質,測量此配對融合蛋白質之螢光壽命,嘗試此分析平台的建立。當同時表現CesT-Cherry與Tir-GFP於大腸桿菌中,此系統確實可偵測出供體壽命縮減,卻也測得可能是不完整螢光蛋白質導致之干擾曲線。整體來說,的確有供體螢光壽命下降的現象,可推測細菌內部此配對蛋白質確實有交互結合。儘管仍須進一步調適實驗條件,本研究初步已成功建立了應用於細菌內分子之FLIM分析系統。
Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a pathogen causes hemorrhagic diarrhea and hemolytic-uremic syndrome, employs the type III secretion system to deliver proteins into host cells. Mechanistically, a pedestal-like structure is formed between the bacterium and the host cell, then a tight bacterial attachment is achieved. These characteristics have been attributed to a pathogenic island on the bacterial chromosome known as the locus for enterocyte effacement(LEE), a cluster of 41 predicted open reading frame(ORFs), of which some have been characterized. Among the uncharacterized ORFs, l0038 encoded a protein of 152 amino acid residue with a predicted pI of 5.5. An l0038–deleted strain created by one-step inactivation of chromosomal gene gave decrease of EspA synthesis and lost most of secrected proteins, suggested that L0038 may act as one of the TTSS components.Unfortunately, there is no way to complement the secretion phenotype of the deleted strain byexogenously expressing L0038, so the hypothesis might need further investigation. In this study, the interaction between L0038 and EscU, and also that between EscU and L0050 were demonstrated by bacterial two-hybrid assay. The sequential interactions between EspA, L0050, EscU and L0038 may imply the decrease of EspA in l0038 mutant may be due to the fail in recruitment of EspA to TTSS machinery. The protein interactions indeed play an essential role in building the TTSS and in secreting the TTS proteins. Accordingly, we apply the technology of FRET/FLIM in in vivo analyzing the interactions between bacterial proteins. Co-expression of both fluorescence-tags fused CesT and Tir in JM109 was used as positive control to set up the FLIM assay in bacteria. Although the possible interference of non-intact fusion protein ruined the analysis result, the shorter life time of donor fluorescence as comparing with that with donor fluorescence protein only demonstrated that there is an in vivo protein-protein interaction between CesT and Tir. Results clearly showed the success in applying FLIM assay in analysis of bacterial samples.
中文摘要 3
Abstract 5
第一章 緒論 7
1. 大腸桿菌概論 7
2. 腸出血性大腸桿菌概論 (Enterohemorrhagic E. coli, EHEC) 8
3. 出血性大腸桿菌之致病機制 8
4. 第三型分泌系統 (type III secretion system; TTSS) 10
5. LEE致病島嶼 (the locus of enterocyte effacement island, LEE island) 11
6. L0038先前之研究 12
7. 目前探討蛋白質交互作用之生物化學研究方法 13
8. 螢光原理與特性 14
9. 螢光共振能量轉移(Förster or fluorescence resonance energy transfer; FRET) 15
10. 螢光壽命呈像顯微術(fluorescence lifetime imaging microscopy; FLIM) 17
11. Tir與其侍衛蛋白質CesT (Tir and the chaperone of Tir, CesT) 18
12. 本文動機與方向 19
第二章 材料與方法 21
1. 細菌菌株 (bacterial strain) 21
2. 質體與引子 (plasmids and primers) 21
3. 抗體 (antibodies) 21
4. 細菌的培養 (bacterial culture) 21
5. 細菌質體萃取與純化 (bacterial plasmid extraction) 22
6. 聚合酶連鎖反應 (polymerase chain reaction; PCR) 22
7. 瓊脂凝膠製作及電泳 (agarose gel electrophoresis) 23
8. 質體構築 (plasmid construction) 23
9. 勝任細胞製備 (Competent cell preparation) 24
10. 細菌轉型 (Transformation) 25
11. 蛋白質之過量表現 (Protein expression) 25
12. SDS聚丙烯醯胺凝膠電泳 (SDS-PAGE) 26
13. 西方墨點法 (Western blotting) 26
14. 鎳離子管柱純化(nickel ion-column purification) 27
15. 三氯醋酸沉澱 (TCA precipitation) 28
16. 細菌雙雜合試驗 (bacterial two-hybrid system) 29
17. β-半乳糖苷酶活性測試 (β-galactosidase activity assay; Miller Assay) 29
18. 染色體基因一步失活法(One-step inactivation of chromosomal gene) 30
19. 螢光壽命顯微術(FLIM)之架構 31
第三章 結果 33
1. l0038基因刪除突變株之構築及篩選 33
2. l0038基因刪除突變株對第三型分泌蛋白質合成與分泌之影響 33
3. 利用細菌雙雜合系統,證明L0038與EscU以及EscU與L0050之交互作用 34
4. 構築各表現螢光蛋白質之質體 35
5. 以西方點墨法確認各融合蛋白質最佳表現量之時間點 36
6. 以螢光顯微鏡觀察各螢光蛋白質最佳表現量之時間點 36
7. 透過Ni2+親合性管柱純化確認CesT與Tir之螢光融合蛋白之間仍具有交互作用 37
8. 螢光壽命呈像顯微術(FLIM)接收的訊號 37
9. 實驗樣本之供體螢光(Tir-GFP)壽命降低 38
10. 實驗組與對照組螢光壽命的比較 38
第四章 討論 40
參考文獻 46
附錄 63
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