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研究生:高紹軒
研究生(外文):Shao-Hsuan Kao
論文名稱:大蒜與百慕達草花粉過敏原之鑑定與其免疫性質研究
論文名稱(外文):Identification and immunologic characterization of allergens from garlic and Bermuda grass pollen.
指導教授:周綠蘋周綠蘋引用關係
指導教授(外文):Lu-Ping Chow
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:136
中文關鍵詞:大蒜免疫學百慕達草花粉過敏原蛋白體學蛋白純化
外文關鍵詞:garlicBermuda grass pollenimmunologyallergenproteomicsprotein purification
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過敏性疾病在近二十年間盛行率日漸增加,已成為現代人最為困擾的疾病之一,其症狀從皮膚輕微的紅、腫、癢,一直到氣喘、鼻炎、異位性皮膚炎,甚至是具有生命威脅的全身性過敏以及過敏性休克等等,其中以第一型過敏 (Type I hypersensitivity)最具危急性。因此,本研究的主要目標為造成第一型過敏疾病之重要過敏原,進行其鑑定與生化免疫特性之分析與研究。
本研究分兩大部分,分別針對大蒜與百慕達草花粉這兩種高盛行率的致敏物種,進行其重要過敏原的鑑定與分析。利用蛋白體學結合免疫學的實驗設計,以二維電泳將複雜的粗蛋白萃取物進行分離,經由過敏病人血清進行IgE結合蛋白的偵測,再利用In-gel digestion、MALDI-TOF-MS、LC tandem MS/MS質譜分析以及資料庫搜尋比對、N端蛋白質定序、胺基酸序列分析與比對、專一性抗血清辨識以及酵素活性分析等多重鑑定程序,完成各個重要過敏原的鑑定。
第一部份:首次鑑定出蒜氨酸酶為大蒜之重要過敏原,並完成蒜氨酸酶的純化以及抗血清誘發 (antiserum induction)與臨床皮膚測試 (intradermal skin test) 等免疫性質之分析。此外,本研究透過物種間的交叉辨識反應 (cross-reactivity)、IgE結合之抑制 (IgE inhibition)與過碘酸氧化實驗,證實蒜氨酸酶在百合科植物如蒜、紅蔥與洋蔥中,具有與大蒜蒜氨酸酶之IgE結合性交叉反應,以及蒜氨酸酶之糖基分子對於其IgE結合性扮演重要之角色等性質。
第二部份:首次利用「次蛋白體」與多重鑑定程序的實驗設計,進行百慕達草花粉的過敏原蛋白體分析,並鑑定出六種新的IgE結合蛋白,分別是enolase 2、malate dehydrogenase、elongation factor 2、aldolase、pathogenesis-related protein以及Phl p 11 homologue。其中,enolase 2在本研究中的IgE辨識率高達90%以上,已正式登錄並命名為Cyn d 22。本研究亦進一步完成Cyn d 22之純化、選殖 (cDNA cloning),酵素活性分析、圓二色光譜分析,以及物種間的交叉辨識反應、IgE結合之抑制反應、碘酸氧化實驗、刺激細胞增生 (proliferation)與組織胺釋放 (histamine release)、細誘發胞激素之產生(cytokine production)等生化與免疫特性分析。
綜合本研究之實驗結果,說明蛋白體學結合免疫學的概念與實驗設計,對於過敏原的鑑定與特性分析是一套精確、完整且高效率的鑑定分析模式。完成鑑定的重要過敏原,不僅對其純化與性質分析有很大的幫助,在過敏原基因的選殖上也提供了重要的資訊。進一步的免疫特性研究,除證實與分析過敏原的致敏能力之外,亦可推測其可能的致敏機制。因此,精確與高效能的過敏原鑑定與分析,對於未來的過敏診斷、免疫治療以及相關的藥物發展為首要的關鍵之一。
Hypersensitivity is one of the most irritating diseases. It results in atopic disorders, such as urticaria, edema, dermatitis, rhinitis and asthma. And sometimes, lethal systematic anaphylaxis and allergic shock will also occur. Among the allergic diseases, type I hypersensitivty mediated by IgE is the most acute and suffering. Here, we focused on the identification and immunologic characterization of these important allergens triggering type I hypersensitivity.
First, we identified and characterized alliin lyase as the important allergen from garlic (Allium sativum). We have purified the allergenic alliin lyase, and investigated the allergen by using periodate-oxidation assay, IgE-crossreactivity assay, IgE-inhibition assay and intradermal skin test. In this study, we have demonstrated that the garlic allin lyase triggered type I hypersensitity, presented IgE-crossreactivity and IgE-inhibition with the enzymes from shallot, leek and onion, and showed weaker IgE-binding after its carbohydrates oxidized.
Second, we established the 2-D protein profile and 2-D IgE-reactive profiles of Bermuda grass pollen (BGP) for an overview of allergenic proteins. By using our newly designed “major allergen depletion approach” and “multiple identification process”, an protein identification process combining of N-terminus homology, peptde-mass fingerprinting, peptide sequence identification, enzyme activity assay and specific Abs recognition, we have successfully identified six novel IgE-reactive proteins from BGP, they are enolase 2, aldolase, elongation factor 2, malate dehydrogenase, pathogenesis-related protein 1, and Phl p 11 homologues. The allergenic enolase 2 with high prevalence was formly named as Cyn d 22 and further investigated. We have cloned and purified the recombinant Cyn d 22, and also demonstrated that native Cyn d 22 could tirgger histamine release, PBMC proliferation, cytokine production, and showed IgE-crossreactivity and IgE-inhibition with enolase from S. cerevisiae.
This is the first time to identify and characterize garlic and BGP allergens by a combination of proteomic and immunologic approaches. This strategy provides an efficient and useful tool for the identification and characterization of novel allergens and should greatly improve the studies in allergy sensitization. Of significance, it may also provide important information on the development of a new class of biopharmaceuticals and future application of diagnoses, desensitizing treatments and immunotherapies for allergic disease.
謝誌 (Acknowledge) i
中文摘要 v
英文摘要 (Abstract) vi
表圖目錄 (List of Tables and figures) vii
縮寫表 (Abbreviations) ix
1. 研究背景、目標與實驗設計 1
1.1 過敏疾病與其致病機制 2
1.1.1 過敏疾病之簡介與流行病學調查 2
1.1.2 過敏疾病之致病機制 5
1.2 現階段的過敏原研究概況 7
1.3 研究目的與實驗設計 9
1.4 蛋白體學實驗方法 12
1.4.1 儀器及裝置 12
1.4.2 藥品與試劑 13
1.4.3 實驗方法 13
1.5 免疫學實驗方法 22
1.5.1 儀器與裝置 22
1.5.2 藥品與試劑 22
1.5.3 臨床試驗對象 23
1.5.4 實驗動物 23
1.5.5 實驗方法 23
2. 以蛋白體學法鑑定分析大蒜過敏原,並探討其免疫特性 30
2.1 動機與目的 30
2.2 實驗材料與方法 32
2.2.1 實驗材料 32
2.2.2 大蒜過敏原之純化 32
2.3 實驗結果 34
2.3.1 大蒜過敏原之分析與鑑定 34
2.3.2 大蒜過敏原之純化 35
2.3.3 大蒜過敏原之免疫特性分析 36
2.4 討論 39
2.4.1 大蒜過敏原之重要性與致敏機制 39
2.4.2 大蒜過敏原的鑑定 40
2.4.3 蒜氨酸酶過敏原之免疫特性 40
2.5 表圖與說明 (詳見表圖目錄) 42
3. 百慕達草花粉IgE結合蛋白之蛋白體學研究及過敏原Cyn d 22的鑑定、
選殖與免疫特性分析 51
3.1 動機與目的 51
3.2 實驗材料與方法 53
3.2.1 儀器裝置 53
3.2.2 實驗材料與試劑 53
3.2.3 百慕達草花粉IgE結合蛋白之蛋白體學研究 54
3.2.4 百慕達草花粉過敏原 Cyn d 22之純化 55
3.2.5 百慕達草花粉過敏原rCyn d 22之選殖 57
3.2.6 百慕達草花粉過敏原重組蛋白 rCyn d 22之表現與純化 60
3.2.7 百慕達草花粉過敏原Cyn d 22之生化性質分析 62
3.3 實驗結果 64
3.3.1 百慕達草花粉IgE結合蛋白之蛋白體研究 64
3.3.2 百慕達草花粉過敏原nCyn d 22之純化 68
3.3.3 百慕達草花粉重組過敏原rCyn d 22之選殖與序列比對 68
3.3.4 百慕達草花粉過敏原重組蛋白rCyn d 22之表現與純化 69
3.3.5 百慕達草花粉過敏原Cyn d 22之生化性質分析 70
3.3.6 百慕達草花粉過敏原Cyn d 22之免疫特性分析 71
3.4 討論 74
3.4.1 花粉過敏原的重要性 74
3.4.2 百慕達草花粉過敏原的分析與鑑定 75
3.4.3 百慕達草花粉過敏原Cyn d 22的序列分析 76
3.4.4 自然態nCyn d 22與重組蛋白rCyn d 22之生化特性比較 77
3.4.5 自然態nCyn d 22與重組蛋白rCyn d 22之免疫特性比較 77
3.4.6 自然態nCyn d 22之免疫特性分析 78
3.5 圖表與說明 (詳見圖表目錄) 80



4. 總結與未來展望 110
4.1 過敏治療與抗過敏藥物之研發 110
4.2 免疫蛋白體學 113
4.3 過敏機制之研究 115
4.4 過敏研究之未來展望 117
參考文獻 119
個人簡歷VITA 135
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