主題一:
異位性疾病諸如過敏性鼻炎、皮膚炎與氣喘的盛行率在已開發國家中具有顯著增加的趨勢。然而長久以來，許多人類的過敏性疾病已經被證實和吸入徽菌的孢子有關。據統計，大約有20 % 到30 %的異位性病人會對黴菌產生過敏症狀；而6 %的一般個體也會對黴菌過敏。
已知點青黴菌 (Penicillium notatum) 是一種常見的室內性黴菌，同時也是導致呼吸性過敏的最主要來源之一。依據免疫轉漬與蛋白質N 端定序的研究分析，得知青黴菌 (Penicillium) 與麴黴菌 (Aspergillus) 這兩屬黴菌的主要過敏原蛋白質是鹼性絲胺酸蛋白質水解酶 (alkaline serine proteases ) 或是液泡內絲胺酸蛋白質水解酶 (vacuolar serine proteases)。本實驗係利用會辨識橘青黴菌鹼性絲胺酸水解酶的單株抗體 ”G11A10” 進行免疫二次元電泳後，將標的蛋白質以in-gel digestion技術切割成小的 peptide片段，接著進行MALDI-TOF質譜分析，得知其peptide map而鑑定到一個點青黴菌的主要過敏原，命名為Pen n 18。進一步地，藉由分子生物得技術完成Pen n 18基因選殖的工作，並依照cDNA序列推演其相對應的蛋白質序列。由於依據cDNA所推演出來的Pen n 18共有494個胺基酸，這與實際上所見的成熟蛋白質分子量( 37 kDa) 並不一致，故推測這之間的差異是由位在蛋白質N 端與C 端的前序列 (prosequences) 所造成。經由蛋白質的序列比對分析，發現Pen n 18與來自其它種類黴菌的液泡內絲胺酸蛋白質水解酶之間具有相當高的序列相似度。利用大腸桿菌M15 pQE30表現系統大量製造出帶有His標籤的重組蛋白質，再以親和力管柱純化重組蛋白質。該純化後的重組蛋白質不但具有辨識病人IgE的能力，同時以抑制試驗得知Pen n 18與來自橘青黴菌的主要過敏原 ”Pen c 18”之間有高度交叉反應，暗示這兩個分子具有相同的IgE結合決定點。未來，該重組過敏原或許可應用於診斷和免疫療法上。
主題二:
過敏性疾病在全世界到處可見，然而在最近的這二十年間，其盛行率是日漸增加。依據世界各個地理區域的相關報導，均指出因接觸到暴露在空氣中的黴菌過敏原而罹患呼吸性過敏疾病是經常可見的。同時青黴菌 (Penicillium) 與麴黴菌 (Aspergillus) 黴菌已被鑑定為會引發外生性支氣管性氣喘疾病的室內型常見之黴菌菌屬。
黃麴黴菌 (Aspergillus flavus) 與黑色麴黴菌 (Aspergillus niger) 是大台北地區患有異位性疾病的兒童，最主要接觸到的室內型黴菌菌種，其出現頻率大於 50 %。利用會對黴菌過敏的病人混和血清進行二次元免疫電泳分析而鑑定到一個重要的黃麴黴菌過敏原” Asp fl 1”。 藉由分子選殖技術將該過敏原的cDNA選殖出並完成其序列分析。全長的cDNA是一段載有403個胺基酸，分子量為 42 kDa 的原生蛋白質 (precusor) 序列。經由切割掉由21個胺基酸所組成的putative signal peptide和100個胺基酸構成的propeptide後，形成一個分子量為 33 kDa，等電點為 6.3，共包含282個胺基酸的成熟蛋白質。分析比對該 Asp fl 1蛋白質序列後發現其與來自細菌的過敏原: subtilisin、橘青黴菌 (Penicillium citrium) 過敏原：Pen c13以及煙色麴黴菌 (Aspergillus fumigatus) 的毒性因子存在有 27 ％ 至84 ％ 的序列相同度。利用載體PQE-30於大腸桿菌M15菌株表現系統，大量製造出重組蛋白質 (rAsp fl 1)，經由親和力管柱純化的重組Asp fl 1具有辨識對麴黴菌過敏的病人血清IgE之能力。此外由抑制試驗得知該重組蛋白質 (rAsp fl 1) 與天然型的Asp fl 1 ( nAsp fl 1) 或是Pen c1之間具有很強的交叉反應，推測黃麴黴菌與橘青黴菌這兩種黴菌應該是擁有共同的IgE決定點的。

Topic I:
Atopic disorders, such as allergic rhinitis, dermatitis and asthma, are increasingly prevalent in the developed countries. It has long been recognized that inhalation of fungal spores are associated with a number of allergic diseases in human. The prevalence of respiratory allergy to fungi is estimated at 20 to 30 % among atopic individuals and up to 6 % in the general population.
Penicillium notatum is a well-known indoor airborne mold and a major source of fungal inhalant allergens. According to the immunoblotting and N-terminal amino acid sequence analysis, it has been reported that the alkaline and/or vacuolar serine proteases are the major allergens in Penicillium and Aspergillus species. In this study, a major allergen from P. notatum, Pen n 18, was identified by two-dimensional immunoblotting using monoclonal antibody G11A10, raised against the vacuolar serine protease of P. citrinum, followed by MALDI-TOF MS analysis of the peptide digest. Pen n 18 was then cloned and the amino acid sequence deduced from the cDNA sequence. The cDNA coded for a 494 amino acid protein, considerably larger than mature Pen n 18, the differences being due to the N-and C-terminal prosequences. The deduced amino acid sequence showed extensive similarity with those of vacuolar serine proteases from various fungi. The Pen n 18 coding sequence was expressed in E. coli as a His-tag fusion protein and purified by Ni2+-chelate affinity chromatography. On immunoblots, the purified recombinant protein specifically bound IgE from mold allergic patients, and cross-inhibition assays demonstrated the presence of common IgE-binding epitopes on Pen n 18 and a major allergen of P. citrinum, Pen c 18. In the future, this recombinant allergen may be used in clinical diagnosis and immunotherapy.
Topic II:
Allergic diseases occur worldwide and their prevalence appears to have been on the increase in the last 2 decades. Reports from different geographical areas have shown that sensitization to moulds through exposure to airborne fungal allergens are common among patients to respiratory allergic diseases. In addition, Penicillium and Aspergillus species have been identified as prevalent indoor airborne fungi that are associated with extrinsic bronchial asthma.
Aspergillus flavus and Aspergillus niger are predominant (> 50 % occurrence frequency) airborne fungi in the residences of atopic children in the Taipei area. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with serum pool of allergic patients on two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full length protein of 403 amino acid precursor of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. The degree of identity were found in a range of 27 % to 84 % among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1 and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.

點青黴菌與黃麴黴菌主要過敏原絲胺酸蛋白水解酶的鑑定，選殖與免疫特性分析------------------------------------------------------------------------------------------------------------1
緒論--------------------------------------------------------------------------2
第一節 前言-------------------------------------------------------------------2
第二節 過敏機制---------------------------------------------------------------2
第三節 過敏的因素-------------------------------------------------------------3
第四節 過敏原的種類-----------------------------------------------------------4
第五節 黴菌過敏原-------------------------------------------------------------4
第六節 過敏疾病的治療策略-----------------------------------------------------5
主題一：點青黴菌主要過敏原絲胺酸蛋白質水解酶 Pen n 18的選殖與免疫特性分析----------9
中文摘要---------------------------------------------------------------------10
英文摘要---------------------------------------------------------------------11
第一章 動機與目的------------------------------------------------------------12
實驗方法---------------------------------------------------------------------13
第一節 黴菌過敏原之鑑定------------------------------------------------------13
第二節 點青黴菌過敏原之選殖--------------------------------------------------15
第三節 點青黴菌過敏原之表現--------------------------------------------------19
第四節 點青黴菌過敏原之免疫特性分析------------------------------------------20
第三章實驗結果---------------------------------------------------------------21
第一節 點青黴菌過敏原之鑑定--------------------------------------------------21
第二節 點青黴菌過敏原之選殖與序列比對----------------------------------------22
第三節 點青黴菌過敏原之表現--------------------------------------------------23
第四節 點青黴菌過敏原之免疫特性分析------------------------------------------24
第四章 討論------------------------------------------------------------------25
第一節 點青黴菌的重要性------------------------------------------------------25
第二節 點青黴菌過敏原的鑑定--------------------------------------------------25
第三節 Pen n 18 序列的分析---------------------------------------------------26
第四節 重組過敏原-rPen n 18與其免疫交叉反應----------------------------------26
第五節 Pen n 18 的B 細胞抗原決定點-------------------------------------------26
第六節 未來展望--------------------------------------------------------------27
第五章 參考文獻--------------------------------------------------------------29
第六章 圖 表-----------------------------------------------------------------35
主題二：黃麴黴菌新的重要過敏原絲胺酸蛋白質水解酵素Asp fl 1之鑑定與特性分析--------50
中文摘要---------------------------------------------------------------------51
英文摘要---------------------------------------------------------------------52
第一章 動機與目的------------------------------------------------------------53
第二章 實驗方法--------------------------------------------------------------54
第一節 黃麴黴菌過敏原之鑑定--------------------------------------------------54
第二節 黃麴黴菌過敏原之選殖--------------------------------------------------55
第三節 黃麴黴菌過敏原之表現--------------------------------------------------57
第四節 黃麴黴菌過敏原之免疫特性分析------------------------------------------58
第三章 實驗結果--------------------------------------------------------------60
第一節 黃麴黴菌過敏原之鑑定--------------------------------------------------60
第二節 黃麴黴菌過敏原之選殖與序列比對----------------------------------------60
第三節 黃麴黴菌過敏原之表現--------------------------------------------------62
第四節 黃麴黴菌過敏原之免疫特性分析------------------------------------------62
第四章 討論------------------------------------------------------------------63
第一節 黃麴黴菌的重要性------------------------------------------------------63
第二節 黃麴黴菌過敏原的鑑定--------------------------------------------------63
第三節 Asp fl 13序列的分析---------------------------------------------------64
第四節 重組過敏原- rAsp fl 其免疫交叉反應-------------------------------------64
第五節 黃麴黴菌鹼性絲胺酸水解酶的分類與演化----------------------------------65
第六節 過敏原生物活性與致敏性------------------------------------------------65
第七節 未來展望--------------------------------------------------------------66
第五章 參考文獻--------------------------------------------------------------67
第六章 圖 表-----------------------------------------------------------------71
發表著作 -------------------------------------------------------------------------------------------------------79

Topic I:
1. Clifford, R. D., Howell, J. B., Radford, M., and Holgate, S. T. (1989) Associations between respiratory symptoms, bronchial response to methacholine and atopy in two age groups of school children. Arch. Dis. Child. 64, 1133—1139
2. Wuthrich, B. (1989) Epidemiology of the allergic diseases: are they really on the increase? Int. Arch. Allergy Appl. Immunol. 90, 3-10
3. 氣喘照護手冊, 行政院環境保護署, 1996.
4. Janeway, C. A., Travers, P., Walport, M., and Capra, J. D. (1999) Immunobiology: The immune system in health and disease. 4th ed., p462-488
5. Barnes, P. J. (1996) Pathophysiology of asthma. Br. J. Clin. Pharmacol. 42, 3-10
6. Anderson, G. P., and Coyle, A. J. (1994) Th2 and ‘Th2-like’ cells in allergy and asthma: Pharmacological perspectives. Trends. Pharmacol. Sci. 15, 324-332
7. Scharenberg, A. M., and Kinet, J. P. (1994) Initial events in FcεRI signal transduction. J. Allergy Clin. Immunol. 94, 1142-1146
8. Marsh, D.G., Neely, J. D., and Breazeale, D. R. et al. (1994) Linkage analysis of IL-4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations. Science 264, 1152—5
9. Baldini, M., Lohman, I. C., Halonen, M., Erickson, R. P., Holt, P. G., and Martinez, F. D. (1999) A polymorphism in the 5’-flanking region of the CD14 gene is associated with circulating soluble CD14 levels with total serum IgE. Am. J. Respir. Cell Mol. Biol. 20, 976—983
10. Marsh, D. G., Hsu, S. H., and Roebber, M. et al. (1982) HLA-Dw2: a genetic marker for human immune response to short ragweed pollen allergen Ra 5 I Response resulting primarily from natural antigenic exposure. J. Exp. Med. 155, 1439—1451
11. Liebers, V., Sander, I., Van Kampen, V., Raulf-Heimsoth, M., Rozynek, P., and Baur, X. (1996) Overview on denominated allergens. Clin. Exp. Allergy. 26, 494-516
12. Latge, J. P., and Paris, S. (1991) The fungal spore and disease initiaton in plants and animals, in: Cole G.T., Moch H.C. (Eds.), Plenum Press, New York, p. 379—401
13. Portnoy, J., Chapman, J., Burge, H., Muieleberg, M., and Solomon, W. (1987) Epicoccum allergy: skin reaction patterns and spore/mycelium disparities recognized by IgG and IgE ELISA inhibition. Ann. Allergy 59, 39—43
14. Burge, H. A. (1989) Airborne-allergenic fungi. Immunol Allergy Clin. North Am. 9, 307—319
15. Kurup, V. P., Fink, J. N. (1993) Fungal Infection and Immunity Responses, in: Murphy, J. W., Friedman, H., Bendinelli, M. (Eds.), Plenum Press, New York, p. 393—404
16. Kurup, V. P. (1989) Hypersensitivity pneumonitis due to sensitization with thermophilic actinomycetes. Immunol. Allergy Clin. North Am. 9, 285—306
17. Kurup, V. P., and Kumar, A. (1991) Immunodiagnosis of aspergillosis. Clin. Microbiol. Rev. 4, 439 —459
18. Latge, J. P., and Paris, S. (1991) The fungal spore and disease initiaton in plants and animals, in: Cole G.T., Moch, H.C. (Eds.), Plenum Press, New York p. 379—401
19. Achatz, G., Oberkofler, H., Lechenauer, E., Simon, B., Unger, A., Kandler, D., Ebner, C., Prillinger, H., Kraft, D., and Breitenbach, M. (1995) Molecular cloning of major and minor allergens of Alternaria alternata and Cladosporium herbarum. Mol. Immunol. 32, 213—227
20. Vijay, H. M., Thaker, A. J., Banerjee, B., and Kurup, V. P. (1999) Allergens and Allergen Immunotherapy, in: Lockey R.F., Bukantz S.C. (Eds.), Marcel Dekker, Inc., New York, p. 133—154
21. Janeway, C. A., and Travers, P. (1997) Allergy and hypersensitivity, in Immunobiology : the immune system in health and disease, 3rd ed. Current Biology ; Garland Publisher, New York
22. Barnes, P. J. (1992) Asthma in adults, in Current therapy in allergy, immunology, and rheumatology., in Lichtenstein L.M., Fauci A.S. (Eds), Mosby, St. Louis, MO, 4th ed. p. 25-32
23. Greenberger, P. A. (1997) Asthma, in Allergic diseases : Diagnosis and Management, in Patterson R., Grammer C.L., Greenberger P.A., (Eds), Lippincott-Raven, New York, 5th ed. p.505-517
24. Shapiro, G. G. (1992) Childhood asthma, in Current therapy in allergy, immunology, and rheumatology. 4th ed. p. 33-38, (Lichtenstein L.M., Fauci A.S. eds.), Mosby, St. Louis, MO
25. Valenta, R., Almo, S., Ball, T., Dolecek, C., Steinberger, P., Laffer, S., Eibensteiner, P., Flicker, S., Vrtala, S., Spitauer, S., Valent, P., Denepoux, S., Kraft, D., Banchereau, J. and Lebecque, S. (1998) The immunoglobulin E-allergen interaction: a target for therapy of type I allergic diseases. Int. Arch. Allergy Immunol. 116, 167-176
26. Chretien, I., Pene, J., Briere, F., De Waal Malefijt, R., Rousset, F., De Vries, J. E. (1990) Regulation of human IgE synthesis. Eur. J. Immunol. 20, 243-251
27. Thompson, R. A., and Kumararatne, D. S. (1989) Hyper-IgE syndrome and H2-receptor blockade. Lancet 2, 630
28. Creticos, P. S. (1992) Immunotherapy with allergens. J. Am. Med. Assoc. 268, 2834-2839
29. Bousquet, J., Lockey, R., Malling, H. J, Alvarez-Cuesta, E., Canonica, G. W, Chapman, M. D., Creticos, P. J, Dayer, J. M., Durham, S. R., Demoly, P., Goldstein, R. J., Ishikawa, T., Ito, K., Kraft, D., Lambert, P. H., Lowenstein, H., Muller, U., Norman, P. S., Reisman, R. E., Valenta, R., Valovirta, E., and Yssel, H.(1998) Allergen immunotherapy: therapeutic vaccines for allergic diseases. Ann. Allergy Asthma Immunol. 81, 401-405
30. Durham, S. R., Walker, S. M., Varga, E. M., Jacobson, M. R., O’Brien, F., Noble, W., Till, S. J., Hamid, Q. A., and Nouri-Aria, K. T. (1999) Long-term clinical efficacy of grass-pollen immunotherapy. N. Engl. J. Med. 34, 468-475
31. La, Rosa. M., Ranno, C., Andre, C., Carat, F., Tosca, M. A., and Canonica, G. W. (1999) Double-blind placebo-controlled evalution of sublingual-swallow immunotherapy with standardized Parietaria judaica extract in children with allergic rhinoconjunctivitis. J. Allergy. Clin. Immunol. 104, 425-432
32. Passalacqua, G., Albano, M., Riccio, A., Fregonese, L., Puccinelli, P., Parmiani, S., and Canonica, G. W. (1999) Clinical and immunologic effects of a rush sublingual immunotherapy to Parietaria species: A double-blind, placebo-controlled trial. J. Allergy ClinImmunol. 104, 964-968
33. Raz, E., Tighe, H., Sato, Y., Corr, M., Dudler, J. A., Roman, R., Swain, S. L., Spiegelberg, H. L., and Carson, D. A. (1999) Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization. Proc. Natl. Acad. Sci. USA 93, 5141-5145
34. Hsu, C. H., Chua, K. Y., Tao, M. H., Lai, Y. L., Wu, H. D., Huang, S. K., and Hsieh, K.H. (1996) Immunoprophylaxis of allergen-induced immunoglobulin E synthesis and airway hyperresponsiveness in vivo by genetic immunization. Nature. Med. 2, 540-544
35. Erb, K. J., Holloway, J. W., Sobeck, A., Moll, H., and Le, Gros. G. (1998) Infection of mice with Mycobacterium bovis-bacillus Calmette-Gue´rin (BCG) suppresses allergen-induced airway eosinophilia. J. Exp. Med. 187, 561-569
36. Su, N. Y., Yu, C. J., Shen, H. D., Pan, F. M. and Chow, L. P. (1999) Pen c 1, a novel enzymic allergen protein from Penicillium citrinum. Eur. J. Biochem. 261, 115-123
37. Chow, L. P., Su, N. Y., Yu, C. J., Chiang, B. L. and Shen, H. D. (1999) Identification and expression of Pen c 2, a novel allergen from Penicillium citrinum. Biochem. J. 341, 51-59
38. Shen, H. D., Liaw, S. F., Lin W. L., Ro, L.H., Yang H. L. and Han S. H. (1995) Molecular cloning of cDNA coding for the 68 kDa allergen of Penicillium notatum using MoAbs. Clin. Exp. Allergy 25, 350-356
39. Shen, H. D., Lin, W. L., Tsai, J. J. Liaw, S. F. and Han, S. H. (1996) Allergenic components in three different species of Penicillium: crossreactivity among major allergens. Clin. Exp. Allergy 26, 444-451
40. Chow, L. P., Chiou, S. H., Hsiao M. C., Yu, C. J. and Chiang, B. L. (2000) Characterization of Pen n 13, a major allergen from the mold Penicillium notatum. Biochem. Biophys. Res. Comm. 269, 14-20.
41. Yu, C. J., Chiou, S. H., Lai, W. Y., Chiang, B. L. and Chow, L. P. (1999) Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease. Biochem. Biophys. Res. Comm. 261, 669-675
42. Chou, H., Lin, W. L., Tam, M. F., Wang, S. R., Han, S. H. and Shen, H. D. (1999) Alkaline serine proteinase is a major allergen of Aspergillus flavus, a prevalent airborne Aspergillus species in the Taipei area. Int. Arch. Allergy Immunol. 119, 282-290
43. Shen, H. D., Lin W. L., Tam, M. F., Wang S. R., Tzean S. S., Huang, M. H. and Han, S. H. (1999) Characterization of allergens from Penicillium oxalicum and P. notatum by immunoblotting and N-terminal amino acid sequence analysis. Clin. Exp. Allergy 29, 642-651
44. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H, Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Ana.l Biochem. 150, 76-85
45. Moritz, R. L., Eddes, J. S., Reid, G. E., and Simpson, R. J. (1996) S-pyridylethylation of intact polyacrylamide gels and in situ digestion of electrophoretically separated proteins: a rapid mass spectrometric method for identifying cysteine-containing peptides. Electrophoresis 17, 907-917
46. Shevchenko, A., Wilm, M., Vorm, O. and Mann, M., (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850-858
47. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159
48. Frohman, M. A., Dush, M. K. and Martin, G. R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. (USA) 85, 8998-9002
49. Kyte, J., and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132
50. Frederick, G. D., Rombouts, P. and Buxton, F. P. (1993) Cloning and characterization of pepC, a gene encoding a serine protease from Aspergillus niger. Gene 125, 57-64
51. Moehle, C. M., Tizard, R., Lemmon, S. K., Smart, J. and Jones, E. W. (1987) Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases. Mol. Cell. Biol. 7, 4390-4399
52. Moehle, C. M., Dixon, C. K. and Jones, E. W. (1989) Processing pathway for protease B of Saccharomyces cerevisiae. J. Cell Biol. 108, 309-325
53. Shinde, U., and Inouye, M. (1994) The structural and functional organization of intramolecular chaperones: the N-terminal propeptides which mediate protein folding. J. Biochem. (Tokyo). 115, 629-636
54. Burge, H. A. (1985) Fungus allergens. Clin. Rev. Allergy 3, 319-329
55. Beaumont, F., Kauffman, H. F., Sluiter, H. J. and De Vries, K., (1985) Sequential sampling of fungal air spores inside and outside the homes of mould-sensitive, asthmatic patients: a search for a relationship to obstructive reactions. Ann. Allergy 55, 740-746
56. Kurup, V. P., Shen, H. D., and Banerjee, B. (2000) Respiratory fungal allergy. Microbes. Infect. 2, 1101-1110
57. Hattori, T., Ichihara, S., and Nakamura, K. (1987) Processing of a plant vacuolar protein precursor in vitro. Eur. J. Biochem. 166, 533-53
58. Zhu, X., Ohta, Y., Jordan, F., and Inouye, M. (1989) Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature (London) 339, 483-484
59. Inouye, M. (1991) Intramolecular chaperone: the role of the pro-peptide in protein folding. Enzyme. 45, 314-21
60. Backman, A., Belin, L., Dreborg, S., Halvorsen, R., Malling, H. J. and Weeke, B. (1991) Standardization of allergenic preparations. Allergy 46, 81-84
61. Platts-Mills, T. A. E., and Chapman, M. D. (1991) Allergen standardization. J. Allergy Clin. Immunol. 87, 621-625
62. Hoyne, G., Bourne, T., Kristensen, N., Hetzel, C., and Lamb, J. (1996) From epitopes to peptides to immunotherapy. Clin. Immunol. Immunopathol. 80, Suppl. 23—30
63. Chapman, M. D. (1991) Use of nonstimulatory peptides: a new strategy for immunotherapy? J. Allergy Clin. Immunol. 88, 300-302
64. Ball, T., Vrtala, S., Sperr, W. R., Valent, P., Susani, M., Kraft, D., and Valenta, R. (1994) Isolation of an immunodominant IgE hapten from an epitope expression cDNA library. J. Biol. Chem 269, 28323-28328
65. Yu, C. J., Chen, Y. M., Su, S. N., Forouhar, F., Lee, S. H., and Chow, L. P. (2002) Molecular and immunological characterization and IgE epitope mapping of Pen n 18, a major allergen of Penicillium notatum. Biochem. J. 363, 707-715
66. Siezen, R. J., Vos, W. M., Leunissen, J. A. M. and Dijkstra, B. W. (1991) Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases. Protein Eng. 4, 719-737
67. Berzofsky, J. A. (1985) Intrinsic and extrinsic factors in protein antigenic structure. Science 229, 932-940
Topic II:
1. Licorish, K., Novey, H. S., Kozak, P., Fairshter, R. D., and Wilson, A. F. (1985) Role of Alternaria and Penicillium spores in the pathogenesis of asthma. J. Allergy Clin. Immunol. 76, 819-825
2. Bennett, J. E. (1990) in Principles and practice of infectious disease (Mandell, G. L., Douglas R. G., and Bennett, J. E. Eds.), pp. 1958-1961, Churchill Living-stone, New York.
3. Arruda, L. K., Platts-Mills, T. A., Fox, J. W. and Chapman, M. D. (1990) Aspergillus fumigatus allergen I, a major IgE-binding protein, is a member of the mitogillin family of cytotoxins. J. Exp. Med. 172, 1529-1532
4. Kumar, A., Reddy, L. V., Sochanik, A. and Kurup, V. P. (1993) Isolation and characterization of a recombinant heat shock protein of Aspergillus fumigatus. J. Allergy Clin. Immunol. 91, 1024-1030
5. Horner, W. E., Helbling, A., Salvaggio, J. E. and Lehrer, S. B. (1995) Fungal allergens. Clin. Microbiol. Rev. 8, 161-179
6. Su, N. Y., Yu, C. J., Shen, H. D., Pan, F. M. and Chow, L. P. (1999) Pen c 1, a novel enzymic allergen protein from Penicillium citrinum. Eur. J. Biochem. 261, 115-123
7. Shen, H. D., Lin, W. L., Tsai, J. J. Liaw, S. F. and Han, S. H. (1996) Allergenic components in three different species of Penicillium: crossreactivity among major allergens. Clin. Exp. Allergy 26, 444-451
8. Chow, L. P., Chiou, S. H., Hsiao M. C., Yu, C. J. and Chiang, B. L. (2000) Characterization of Pen n 13, a major allergen from the mold Penicillium notatum. Biochem. Biophys. Res. Comm. 269, 14-20
9. Yu, C. J., Chiou, S. H., Lai, W. Y., Chiang, B. L. and Chow, L. P. (1999) Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease. Biochem. Biophys. Res. Comm. 261, 669-675
10. Chou, H., Lin, W. L., Tam, M. F., Wang, S. R., Han, S. H. and Shen, H. D. (1999) Alkaline serine proteinase is a major allergen of Aspergillus flavus, a prevalent airborne Aspergillus species in the Taipei area. Int. Arch. Allergy Immunol. 119, 282-290
11. Kurup V. P., Shen, H. D. and Banerjee B. (2000) Respiratory fungal allergy. Microbes Infect. 2, 1101-1110
12. Chow, L. P., Fukaya, N., Miyatake, N., Horimoto, K., Sugiura, Y., Tabuchi, K., Ueno, Y. and Tsugita, A. (1995) Resolution of Fusarium sporotrichioides proteins by two-dimensional polyacrylamide gel electrophoresis and identification by sequence Homology comparison in protein data base. J. Biomed. Sci. 2, 343-352
13. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H, Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76-85
14. Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159
15. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. (USA) 85, 8998-9002
16. Tatsumi, H., Ogawa, Y., Murakami, S., Ishida, Y., Murakami, K., Masaki, A., Kawabe, H., Arimura, H., Nakano E., Motai, H. (1989) A full length cDNA clone for the alkaline protease from Aspergillus oryzae: structural analysis and expression in Saccharomyces cerevisiae. Mol. Gen. Genet. 219, 33-38
17. King, T. P, Hoffman, D., Lowenstein, H., Marsh, D.G., Platts-Mills, T. A. E., and Thomas, W. (1994) Allergen nomenclature. WHO/IUIS Allergen Nomenclature Subcommittee. Int. Arch. Allergy. Immunol. 105, 224-233
18. Jaton-Ogay, K., Suter, M., Crameri, R. Falchetto, R., Fatif, A. and Monod, M. (1992) Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus. FEMS Microbiol. Lett. 71, 163-168
19. Wells, J. A., Ferrari, E., Henner, D. J., Estell, D. A., and Chen, E. Y. (1983) Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res. 11, 7911-7925
20. 氣喘照護手冊, 行政院環境保護署, 1996.
21. Wuthrich B. (1989) Epidemiology of the allergic diseases: are they really on the increase? Int. Arch. Allergy Appl. Immunol. 90, 3-10
22. Portnoy, J., Chapman, J., Burge, H., Muieleberg, M. and Solomon, W. (1989) Epicoccum allergy: skin reaction patterns and spore/mycelium disparities recognized by IgG and IgE ELISA inhibition. Ann. Allergy 59, 39—43
23. Zhu, X., Ohta, Y., Jordan, F., and Inouye, M. (1989) Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature (London) 339,483-484
24. Inouye, M. (1991) Intramolecular chaperone: the role of the pro-peptide in protein folding. Enzyme 45,314-321
25. Hattori, T., Ichihara, S. and Nakamura, K. (1987) Processing of a plant vacuolar protein precursor in vitro. Eur. J. Biochem. 166,533-538
26. Mueller, G. A., Benjamin, D. C. and Rule, G. S. (1998) Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies. Biochemistry 37, 12707-12714
27. Platts-Mills, T. A., Wheatley, L. M. and Aalberse, R. C. (1998) Indoor versus outdoor allergens in allergic respiratory disease. Curr. Opin. Immunol. 10, 634-639
28. Shen, H. D., Choo, K.B., Lee, H. H., Hsieh, J. C., Lin, W. L., Lee, W. R., and Han, S. H. (1991) The 40-kilodalton allergen of Candida albicans is an alcohol dehydrogenase: molecular cloning and immunological analysis using monoclonal antibodies. Clin. Exp. Allergy. 21, 675-681
29. Schulz, O., Laing, P., Sewell, H. F. and Shakib, F. (1995) Der p I, a major allergen of the house dust mite, proteolytically cleaves the low-affinity receptor for human IgE (CD23). Eur. J. Immunol. 25, 3191-3194
30. Shakib, F., Schulz, O., and Sewell, H. (1998) A mite subversive: cleavage of CD23 and CD25 by Der p 1 enhances allergenicity. Immunol. Today 19, 313-316
31. Herbert, C. A., King, C. M., Ring, P. C., Holgate, S. T., Stewart, G. A., Thompson, P. J. and Robinson, C. (1995) Augmentation of permeability in the bronchial epithelium by the house dust mite allergen Der p1. Am. J. Res. Cell Mol. Biol. 12, 369-378
32. King, C., Brennan, S., Thompson, P. J. and Stewart, G. A. Dust mite proteolytic allergens induce cytokine release from cultured airway epithelium. J. Immunol. 161, 3645-3651
33. Robinson, B. W., Venaille, T. J., Mendis, A. H. and McAleer, R. Allergens as proteases: an Aspergillus fumigatus proteinase directly induces human epithelial cell detachment. J. Allergy Clin. Immunol. 86, 726-731
34. Hein J. (1990) Unified approach to alignment and phylogenies. Methods Enzymol. 183, 626-645