葛蘭氏陰性菌脂質A之結構研究

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姓名

胡詠甯(Yung-Ning Hu) 查詢紙本館藏

畢業系所

化學學系

論文名稱

葛蘭氏陰性菌脂質A之結構研究
(Stuctural determination of the lipid A from gram-negative bacteria)

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摘要(中)

脂多醣體（Lipopolysaccharide）又被稱為內毒素，是引發先天性免疫反應的啟動因子，也是導致發炎、敗毒病和敗血性休克而造成葛蘭氏陰性菌致死的原因，因而脂多醣體的生物活性中心，亦即脂質A之相關研究極具重要性。針對不會引起敗血性症狀的葛蘭氏陰性菌之脂質A的研究相當有限，然而研究卻發現部份這類型的脂多醣體或是脂質A對於哺乳動物無毒卻可作為大腸桿菌或是其他劇毒性的脂多醣體細菌的抑制劑。
在本研究中，我們利用甲酚加熱萃取得脂多醣體，並且利用電泳分離並以銀染顯色。我們改變傳統的加熱方法而改以微波加熱，使得反應得以加速同時又足以使得脂多醣體水解脂質A且不會有其餘醣殘留。我們更進一步的利用商業上可以購得的大腸桿菌脂質A(Sigma)作為以新方法自行分離出來的大腸桿菌的標準品來確認所發展分離步驟的可行性。而藉由從大腸桿菌分離並分析脂質A的經驗，我們也成功的從非致病性的葛蘭氏陰性菌之嗜甲烷菌中獲得其脂質A，再利用NMR、ESI-MS、MALDI-TOF MS作為工具，我們提出嗜甲烷菌的脂質A的可能結構為以葡萄糖為骨架並具有四個碳鏈的結構。
總而言之，我們發展出一個更加有效率的方法利用微波加熱而從葛蘭氏陰性菌中分離其脂質A。並藉此方法，我們可以得知嗜甲烷菌脂質A的結構。

摘要(英)

Lipopolysaccharide (LPS), often referred as endotoxin, triggers the body’s innate immune response and is responsible for inflammation, sepsis and septic shock leading to fatalities in gram-negative bacteria. Research on the biological activity of LPS, lipid A, is therefore very important. Lipid A structures of gram-negative bacteria that do not induce sepsis-like syndrome in human are less understood. While several of these LPSs or lipid As have been shown to be non-toxic for mammalian, they are important antagonists of E. coli or other toxic LPS containing strains.
In this research, we used hot phenol extraction to obtain LPS and electrophoresis gel with silver stain to visualize LPS. Moreover, we used microwave, instead of conventional method, to hydrolyze the lipid A from LPS in order to accelerate the chemical reaction and to allow sufficient time to remove the sugars on LPS. We took the advantage of commercial available lipid A extract from E. coli (Sigma) as a model of our research to compare with the lipid A isolated by our isolation procedures. By these experiences of isolating and characterizing the lipid A from E. coli? we successfully obtained the lipid A from the non-pathogenic gram-negative bacteria, M. capsulatus, as well. Using NMR, ESI-MS, MALDI-TOF MS, we deduced the possible structure of lipid A from M. capsulatus. The structure consists of a glucose backbone with four acyl chains attached.
In conclusion, we have developed a more efficient method to isolate lipid A from gram-negative bacteria using microwave heating. Model structural tools have been used to determine the structure of isolated lipid A from M. capsulatus.

關鍵字(中)

★ 脂質A
★ 葛蘭氏陰性菌

關鍵字(英)

★ lipid A
★ gram-negative bacteria

論文目次

摘要 I
Abstract II
謝誌 III
Chapter 1. Introduction 1
1-1 Immune system 1
1-2 The pathogens 1
1-3 Endotoxin - Lipopolysaccharide 3
1-4 Biological active component - Lipid A 5
1-5 Compounds that inhibit the biological actions of LPS 10
1-6 The bacteria used in this research: Escherichia coli and Methylococcus capsulatus (Bath) 12
1-7 Extraction of lipopolysaccharide 13
1-8 Microwave degradation, a new method used for LPS degradation to lipid A 13
1-9 Purpose of the research 14
Chapter 2. Experimental Methods 18
2-1 Materials and instrumentation 18
2-2 Isolation and structural determination of lipid A from Escherichia coli 23
2-2-2 The isolation of lipopolysaccharide was ascertained by SDS-PAGE electrophoresis 23
2-2-3 Silver staining 25
2-2-4 Lipid A isolation 25
2-2-5 TLC test 26
2-3-6 MS spectra 26
2-2-7 NMR spectroscopy 27
2-3 The methods of isolation and structural determination of lipid A from Methylococcus capsulatus (Bath) 27
2-3-1 Lipopolysaccharide extraction and isolation of lipid A 27
2-3-2 Fatty acid analysis 27
2-3-3 Sugar analysis 28
Chapter 3. Results and Discussions 29
3-1 Methodology 29
3-1-1 Phenol-water extraction of lipopolysaccharide 29
3-1-2 Silver staining used to visualize lipopolysaccharide 29
3-1-3 The isolation of lipid A by microwave heating 30
3-2 Structural characterization of isolated lipid A from Escherichia coli 33
3-2-1 ESI-MS 33
3-2-2 MALDI-TOF MS 33
3-2-3 NMR 40
3-3 Structural characterization of isolated lipid A from Methylococcus capsulatus (Bath) 44
3-3-1 Lipopolysaccharide extraction 44
3-3-2 ESI-MS 44
3-3-3 Fatty acid analysis of M. capsulatus 48
3-3-4 Sugar analysis of M. capsulatus 48
3-3-5 MALDI-TOF MS 62
3-3-6 NMR 65
Chapter 4. Conclusions 69
References 70

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指導教授

俞聖法、蔡惠旭
(Sheng-Fa Yu、Hui-Hsu Gavin Tsai)

審核日期

2008-1-14

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