臺灣博碩士論文加值系統

當過敏病人接觸到花粉、塵、動物性皮屑及黴菌等過敏原時，他們血液中的免疫球蛋白E就會有不正常的增多反應發生，這些具有特異性的IgE抗體會引發過敏病人有過敏性鼻炎、結膜炎、皮膚炎、氣喘，甚至致死性的休克等臨床症狀的發生。其中的一些過敏病人特別是因為吸入一些徽菌的孢子或菌絲而產生上述的過敏症狀。現已有超過60黴菌發現與過敏有關;其中青黴菌屬(Penicillium)這種著名的室內型黴菌，已知能夠引起嚴重的支氣管氣喘性疾病，在大台北地區青黴菌屬中又以橘青黴(Penicillium citrinum)這一個種系最為常見，且其中一個被命名為 Pen c1 的主要過敏原已被發現具有能夠廣泛與病人 IgE 抗體強力結合的過敏原特性，是一個相當值得研究的過敏原。
鑑定過敏原與 IgE 結合的抗原決定點，為了解過敏反應免疫致病機制的主要工作之一。本實驗的研究目的即是要找出過敏原 Pen c1 的抗原及過敏原決定點，並分析這些 IgE 抗原決定點的免疫生化特性及探討其在 Pen c1 3-D 結構上的立體相關位置。
為了分析這個過敏原的抗原決定點，我們運用了化學和酵素切割法，及免疫轉印技術，並結合質譜及N端蛋白質序列的分析工作來進行。首先，對於小鼠及兔子血清中IgG抗體，一共有至少8段獨立的線性抗原決定點被找到，此外M39、55A、及5-8H等三株小鼠單株抗體也被發現辨識Pen c1上不同的三個區段。我們也運用這項實驗策略分析病人血清中IgE抗體的辨識區段，一共使用了24條涵蓋Pen c1蛋白質全長的胜片段，針對10位過敏病人來進行這項實驗工作，結果發現，幾乎所有受檢病人的IgE都會結合A148~E166及A243~K274這兩段區域。接著，我們對於這兩段Pen c1重要的免疫顯性胜(immunodominant peptides)進行刺激病人T細胞增生及ELISA胜阻斷性(inhibition assay)的研究工作。
再來，藉由競爭性實驗探討這些抗Pen c1的小鼠單株抗體其是否能夠阻礙人類IgE抗體與完整Pen c1的結合能力。結果推測，一些單株抗體與人類IgE抗體辨識了部分相同的區域，且它們可能是過敏原Pen c1的結構性抗原決定點所在。
最後，我們運用現在電腦模擬分子結構的方法，將這些抗原決定點的立體位置呈現出來，並發現到幾乎所有呈陽性反應的胜片段或多或少都包含了一些暴露在分子外圍的區域。
這些分析結果，將幫助我們對於過敏反應起始步驟有著更進一步的了解，未來這些研究結果希望將可被實際地運用到過敏疾病的診斷及治療上。

Allergic patients tend to mount immunoglobulin E responses against allergens that originate from pollen, mites, animal dander and molds. These allergic patients with specific IgE antibodies show severe clinical symptoms (allergic rhinitis, conjunctivitis, dermatitis, asthma and even lethal anaphylactic shock ) upon allergen contact. It has long been recognized that inhalation of fungal spores or mycelial fragments can produce allergic symptoms in susceptible individuals. Over 60 species of fungi are known to be allergenic. Penicillim species are well-known indoor fungi and have been considered as one of the causative agents of extrinsic bronchial asthma. Penicillim citrinum is the most prevalent Penicillium species in Taipei area, and one protein of P. citrinum which consists of 282 amino acid residues has been identified as a major allergen and designed Pen c1. Because of its high affinity of binding to IgE and high prevalence, the allergen from P. citrinum justifies study.
The identification and characterization of the epitopes of fungal allergens that bind IgE is essential in order to understand the immunopathogenic mechanisms involved in hyper sensitivities reactions. In this research , the identification of the allergenic and antigenic determinants of Pen c1, the analysis of immune and biochemical characters on IgE-epitopes of Pen c1 and the location on 3-D structure of Pen c1 are discussed.
In order to analyze the epitope of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least eight different linear epitopes were demonstrated with distinct binding responses of IgG antibodies against murine and rabbit sera. Furthermore, three monoclonal antibodies mAb M39, mAb 55A and mAb 5-8H against Pen c1 recognized the different segments. The strategy for determining IgG epitopes applied in this study was also useful for determining the epitopes of patient's IgE. Each set of 24 peptides, which distributed throughout the length of the Pen c1 protein, was probed individually with serum IgE from 10 different patients. The sera from the all patients recognized multiple epitopes, and two epitopes (A148~E166, A243~K274) were recognized by almost all patients tested. Moreover, we examined the proliferation of T cells and ELISA peptides inhibition assay by these two B cell immunodominant peptides of allergen Pen c1.
Further, to investigate whether mouse monoclonal anti-Pen c1 Abs are able to inhibit the binding of human IgE Abs to Pen c1, competition experiments were performed. Our studies suggested that some mAbs shared their epitopes with human IgE antibodies, and might recognize conformational epitopes of Pen c1.
Finally, the position of each of the immunodominant epitopes on a homology-based molecular modle of Pen c1 was showed. We found that almost all positive peptides contain the exposed regions of the molecule more or less.
These analyses may contribute to a better understanding of the event in the early steps of the allergic response and will therefore be useful for diagnostic and immunotherapeutic approaches to allergic diseases.

目錄
中文摘要
ABSTRACT
縮寫
第一章、導論--------------------------------------------------------------------------------1
第二章、實驗材料-------------------------------------------------------------------------10
第三章、實驗方法-------------------------------------------------------------------------12
第四章、實驗結果-------------------------------------------------------------------------29
第五章、討論-------------------------------------------------------------------------------37
圖表及說明----------------------------------------------------------------------------------43
參考文獻-------------------------------------------------------------------------------------66

1. Altschul SF. Madden TL. Schaffer AA. Zhang J. Zhang Z. Miller W. Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. (1997) 25, 3389-3402.
2. Ball G. Shelton MJ. Walsh BJ. Hill DJ. Hosking CS. Howden ME. A major continuous allergenic epitope of bovine beta-lactoglobulin recognized by human IgE binding. Clin. & Exp. Allergy. (1994) 24, 758-764.
3. Ball T., Vrtala S., Sperr W. R., Valent P., Susani. M., Graft D., Valenta R. Isolation of an immunodominant IgE hapten from an epitope expression cDNA library : dissection of the allergic effector reaction. J. Biol. Chem. (1994) 269 , 28323-28328.
4. Bauer L. Ebner C. Hirschwehr R. Wuthrich B. Pichler C. Fritsch R. Scheiner O. Kraft D. IgE cross-reactivity between birch pollen, mugwort pollen and celery is due to at least three distinct cross-reacting allergens: immunoblot investigation of the birch-mugwort-celery syndrome. Clin. & Exp. Allergy. (1996) 26, 1161-1170.
5. Braden BC, Polhak RJ, Mason TJ, Tribbick G, Schoofs PG. Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods (1987) 102 259-274.
6. Burks A.W., Shin D., Cockrell G., Stanley J.S., Helm R.M. and Bannon G.A. Mapping and mutational analysis of the IgE-binding epitopes on Ara h 1, a legume vicilin protein and a major allergen in peanut hypersensitivity. Eur. J. Biochem. (1997) 245, 334-339.
7. Cardaba B. Del Pozo V. Jurado A. Gallardo S. Cortegano I. Arrieta I. Del Amo A. Tramon P. Florido F. Sastre J. Palomino P. Lahoz C. Olive pollen allergy: searching for immunodominant T-cell epitopes on the Ole e 1 molecule. Clin. & Exp. Allergy. (1998) 28, 413-422.
8. Chang T. W. The pharmacological basis of anti-IgE therapy. Nature Biotech. (2000) 18, 157- 162.
9. Chauhan B., Knutsen A. P., Hutcheson P. S., Slavin R. G., and Bellone C. J. T cell subsets, epitope mapping, and HLA-restriction in patients with allergic bronchopulmonary aspergillosis. J. Clin. Invest. (1996) 97, 2324-2331.
10. Chen Z. , Kampen V. V. , Raulf-heimsoth M. and Baur X. Allergenic and antigenic determinants of latex allergen Hev b1 : peptide mapping of epitopes recognized by human, murine and rabbit antibodies. Clin. & Exp. Allergy. (1996) 26, 406-415.