臺灣博碩士論文加值系統

中文摘要
狗牙根禾花花粉為台灣及世界各地主要的禾花花粉過敏原, 而Cyn d 1是分子量34kDa的主要過敏原成份, 也是本實驗室所研究的重點。本研究室在過去已利用分子選殖的方法得知Cyn d 1可分酸性和鹼性過敏原基因。此外, 利用兩類Cyn d 1重組蛋白在動物實驗也證實兩類Cyn d 1免疫性質確有差異。
本研究進一步分析台灣地區之狗牙根花粉中Cyn d 1屬酸性或鹼性, 並以酵母菌表現的兩類Cyn d 1重組蛋白探討兩類Cyn d 1過敏原性 ( allergenicity ) 之不同。
首先分析台灣地區狗牙根花粉究竟含酸性Cyn d 1或鹼性 Cyn d 1, 以RT-PCR分析在屏東地區所採集到的花粉, 發現只具酸性Cyn d 1之mRNA, 不具鹼性之mRNA, 再用分子選殖的方法得到九株Cyn d 1 cDNA clone, 和國外酸性Cyn d 1序列做比較之後, 發現依據第157 ( S/N ) 、第181 ( P/S )及第192 ( L/Q ) 胺基酸的不同可將酸性Cyn d 1再分成兩群, 台灣地區屬於前者酸性Cyn d 1。
進而評估Cyn d 1重組蛋白的生物性功能 ( biological function ), 首先進行組織胺釋放試驗, 取四個對狗牙根花粉過敏者週邊血進行研究, 發現對於其中三人, 酸性重組蛋白在引發嗜鹼性球釋放出組織胺的能力皆較鹼性重組蛋白強。 且再用免疫競爭抑制法分析哪一種重組蛋白具較強的IgE競爭力, 發現六個病人裡, 有三個待測血清中, 酸性重組蛋白和IgE結合的能力強過鹼性重組蛋白, 其中以酸性同質異構物11號對十五位過敏病人的pool serum有最強的IgE競爭抑制力, 而鹼性同質異構物9-4-3的競爭抑制力則最低。最後用重組蛋白引起試管內淋巴細胞活化反應, 從六位病人中則看到酸鹼重組蛋白皆可刺激淋巴球增生, 刺激能力並無明顯差異。

英文摘要
Bermuda grass ( Cynodon dactylon ) pollen is a major grass pollen allergen found not only in Taiwan but also all over the world. The purpose of this study is to investigate the 34 kilodalton ( kDa ) major allergen of BGP, Cyn d 1. In the past, Cyn d 1 had been separated into the acidic and basic proteins by molecular cloning, and the immunological properties of the two groups of Cyn d 1 were different in the animal model experiment using the two groups of Cyn d 1 recombinant antigens .
In the present study, we further analyzed to which group Taiwan’s Cyn d 1 of BGP belonged. We used the two groups of recombinant Cyn d 1 proteins which were expressed by Yeast Pichia to study the difference of two groups of Cyn d 1.
First, we analyzed whether Taiwan’s BGP contains acidic Cyn d 1 or basic Cyn d 1. We used Reverse Ttranscription - Polymerase Chain Reaction to analyze Ping Tung’s BGP that we took, only mRNA of acidic Cyn d 1 was found but mRNA of basic Cyn d 1 was not. We got nine cDNA clones of Cyn d 1 by molecular cloning. According to the 157th ( S/N )、181th ( P/S ) and 192th ( L/Q ) amino acid substitution we divided acidic Cyn d 1 into two groups after being compared with foreign Cyn d 1, and Taiwan’s Cyn d 1 was the former.
Then we further evaluated the biological function of recombinant Cyn d 1 proteins. First we practiced histamine release assay to investigate peripheral blood of four patients who are sensitive to BGP. The acidic recombinant proteins were found to have better ability in inducing basophiles to release histamine than basic recombinant proteins toward three of the four patient’s peripheral blood. We further analyzed which recombinant protein had higher competitive ability by dot inhibition, and in three of the six patient’s serum, we found the IgE-binding capacity of acidic recombinant proteins were higher than basic recombinant proteins. Among these recombinant proteins toward pool serum, acidic isoform 11 was found to has the highest IgE competitive ability , and the IgE competitive ability of the basic isoform 9-4-3 was the lowest. Finally, we used recombinant proteins to elicit lymphocyte activation of six patients, not much different stimulation capacity between acidic and basic recombinant proteins was found.

目錄
中文摘要 -----------------------------------------------------------Ⅰ
英文摘要 -----------------------------------------------------------Ⅲ
緒論 ----------------------------------------------------------------- 1
材料與方法 -------------------------------------------------------- 11
結果 ----------------------------------------------------------------- 24
討論 ----------------------------------------------------------------- 28
參考文獻 ----------------------------------------------------------- 33
圖表 -----------------------------------------------------------------

參考文獻
韓韶華、謝長富、蔡來誠、莊雨奇。台北地區的風媒植物及其花粉。中華微免1980；13：60-86。
蘇崧南、劉繼昌、李明貞、沈弘德、韓韶華。狗牙根花粉過敏原之分離、純化、與部分性質分析。中華微免1986；19：263-275。
Breitenbach M, Ferreira F, Jilek A, Swoboda I, Ebner C, Hoffmann-Sommergruber K, Briza P, Scheiner O, Kraft D. Biological and immunological importance of Bet v 1 isoforms. Adv Exp Med Biol 1996;409:117-26.
Breitenbach M, Ferreira F, Jilek A, Swoboda I, Ebner C, Hoffmann-Sommergruber K, Briza P, Scheiner O, Kraft D. Biological and immunological importance of Bet v 1 isoforms. Adv Exp Med Biol 1996; 409:117-26.
Chang HN, Lin FM, Chang YF. Correlation of skin test to RAST, and Prist in asthma patients. Clin Med J 1984; 34:376-383.
Chang ZN, Liu CC, Tam MF, Peng HJ, Ysai JJ, Han SH. Characterization of the isoforms of group I allergen of Cynodon dactylon. J Allergy Clin Immunol. 1995; 95(6):1206-1214.
Chang ZN, Tam MF, Liu CC, Chi CW, Peng HJ, Han SH. Using monoclonal antibodies to characterize a sequential epitope on the group I allergen of Bermuda grass pollen. Int Arch Allergy Immunol 1997;114:258-264.
Chang ZN, Peng HJ, Lee WC, Chen TS, Chua KY, Tsai LC, Chi CW, Han SH. Sequence polymorphism of the group 1 allergen of Bermuda grass pollen. Clin Exp Allergy. 1999; 29(4):488-496.
Ebner C, Ferreira F. Hypoallergenic isoform variants of allergens-a new concept for specific immunotherapy. ACI international 1997, 9/3.
Ebner C. Immunological mechanism operative in allergen-specific immunotherapy. 1999 ; 119 (1) Int Arch Allergy Immunol:1-5.
Ford SA, Baldo BA. Identification of Bermuda grass (Cynodon dactylon) -pollon allergens by electroblotting. J Allergy Clin Immunol 1987; 79:711-720.
Ferreira FD, Hoffmann-Sommergruber K, Breiteneder H, Pettenburger K, Ebner C, Sommergruber W, Steiner R, Bohle B, Sperr WR, Valent P. Purification and characterization of recombinant Bet v I, the major birch pollen allergen. Immunological equivalence to natural Bet v I. J Biol Chem 1993 ; 268 (26):19574-80.
Ferreira F, Hirtenlehner K, Jilek A, Godnik-Cvar J, Breiteneder H, Grimm R, Hoffmann-Sommergruber K, Scheiner O, Kraft D, Breitenbach M, Rheinberger HJ, Ebner C. Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy. J Exp Med 1996 ;183 (2):599-609.
Ferreira F, Hirthenlehner K, Briza P, Breiteneder H, Scheiner O, Kraft D, Breitenbach M, Ebner C. Isoforms of atopic allergens with reduced allergenicity but conserved T cell antigenicity: possible use for specific immunotherapy. Int Arch Allergy Immunol 1997 ;113 (1-3):125-7
Fung-Leung W-P, Sousa-Hitzler JD, Ishaque A, Zhou L, Pang J, Ngo K, Panakos JA, Chourmouzis E, Liu F-T, Lau CY. Transgenic mice expressing the human high-affinity immunoglobulin(Ig) E receptor respond to human IgE in mast cell degranulation and in allergic reactions.
Godnic-Cvar J, Susani M, Breiteneder H, Berger A, Havelec L, Waldhor T, Hirschwehr R, Valenta R, Scheiner O, Rudiger H, Kraft D, Ebner C. Recombinant Bet v 1, the major birch pollen allergen, induces hypersensitivity reactions equal to those induced by natural Bet v 1 in the airways of patients allergic to tree pollen. J Allergy Clin Immunol 1997 ; 99 (3):354-9.
Hsu CC,台灣植物誌 1975 ; 375
Han SW, Chang ZN, Chi CW, Perng HJ, Liu CC, Tsai JJ, Tam MF. Use of monoclonal antibodies to isolate and characterize Cyn d 1, the major allergen of Bermuda grass pollen. J Allergy Clin Immunol 1993; 92:549-558.
Hartl A, Kiesslich J, Weiss R, Bernhaupt A, Mostbock S, Scheiblhofer S, Flockner H, Sippl M, Ebner C, Ferreira F, Thalhamer J. Isoforms of the major allergen of birch pollen induce different immune responses after genetic immunization. Int Arch Allergy Immunol. 1999 ;120 (1):17-29.
Jorgen Nedergaard Larsen. Isoallergens-Significance in allergen exposure and response. ACI News, 1995
King TP, Hoffman D,Lowenstein H, Marsh DG, Platts-Mills TAE, Thomas W. Allergen nomenclature. Clin Exp Allergy 1995; 25:27-37.
Kraft D, Ferreira F, Ebner C, Valenta R, Breiteneder H, Susani M, Breitenbach M, Scheiner O. Recombinant allergens: the future of the diagnosis and treatment of atopic allergy. Allergy 1998;53 (45 Suppl):62-6.
Kraft D, Ferreira F, Vrtala S, Breiteneder H, Ebner C, Valenta R, Susani M, Breitenbach M, Scheiner O. The importance of recombinant allergens for diagnosis and therapy of IgE-mediated allergies. Int Arch Allergy Immunol 1999 ; 118 (2-4):171-6.
Menz G, Dolecek C, Schonheit-Kenn U, Ferreira F, Moser M, Schneider T, Suter M, Boltz-Nitulescu G, Ebner C, Kraft D, Valenta R . Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen. Clin Exp Allergy 1996 ;26(1):50-60.
Orren A, Dowdle EB. Studies on Bermuda grass pollen allergens. S Afr Med J 1977; 51:586-591.
Oliver A, Arthur H, reto C, Fatima F, Michael B, Werner JP. Reduced in vivo allergenicity of Bet v 1d isoform, a natural component of birch pollen. J Allergy Clin Immunol 1999; 104:1239-43.
Peter JB. Therapeutic strategies for allergic diseases. Nature 1999; 402:B31-B39.
Renz H. Aerosolized antigen exposure without adjuvant caused increased IgE production and increased airway responsiveness in the mouse. J Allergy Clin Immunol 1992; 89:1127-1138.
Shen HD, Wang SR, Tang RB, Chang ZN, Su SN, Han SH. Identification of allergens and antigens of Bermuda grass (Cynodon dactylon) pollen by immunoblot analysis. Clin Allergy 1988; 18:401-409.
Susani M, Jertschin P, Dolecek C, Sperr WR, Valent P, Ebner C, Kraft D, Valenta R, Scheiner O . High level expression of birch pollen profilin (Bet v 2) in Escherichia coli: purification and characterization of the recombinant allergen. Biochem Biophys Res Commun 1995 Oct 4;215(1):250-63.
Smith PM, Suphioglu C, Griffith IJ, Theriault K, Knox B, Singh MB. Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen. J Allergy Clin Immunol 1996; 98:331-343.
Valenta R, Duchene M, Vrtala S, Birkner T, Ebner C, Hirschwehr R, Breitenbach M, Rumpold H, Scheiner O, Kraft D. Recombinant allergens for immunoblot diagnosis of tree-pollen allergy. J Allergy Clin Immunol 1991 ;88(6):889-94.
Valenta R, Sperr WR, Ferreira F, Valent P, Sillaber C, Tejkl M, Duchene M, Ebner C, Lechner K, Kraft D. Induction of specific histamine release from basophils with purified natural and recombinant birch pollen allergens. J Allergy Clin Immunol 1993 ;91(1 Pt 1):88-97.
Van Neerven RJ, Ebner C, Yssel H, Kapsenberg ML, Lamb JR. T-cell responses to allergens: epitope-specificity and clinical relevance. Immunol Today 1996 ; 17 (11):526-32.
Vrtala S, Hirtenlehner K, Susani M, Hufnagl P, Binder BR, Vangelista L, Pastore A, Sperr WR, Valent P, Ebner C, Kraft D, Valenta R. Genetic engineering of recombinant hypoallergenic oligomers of the major birch pollen allergen, Bet v 1: candidates for specific immunotherapy. Int Arch Allergy Immunol 1999 ;118 (2-4):218-9.
Weiss C, Kramer B, Ebner C, Susani M, Briza P, Hoffmann-Sommergruber K, Breiteneder H, Kraft D, Scheiner O, Breitenbach M, Ferreira F. High-level expression of tree pollen isoallergens in Escherichia coli. Int Arch Allergy Immunol 1996 ;110 (3):282-7.
Wayne R. Thomas, Wendy-Anne Smith, Belinda J. Hales. Allergens-Sequence diversity and T-cell recognition. ACI International, 1998
Xu F, Andrew S, Andrew N, Ke Z, David D-S and Andre N. Interleukin 4 activates a signal transducer and activator of transcription (Stat) protein which interacts with an interferon-γ activation on site-like sequence upstream of the Iε exon in human B cell line. J Clin Invest 1995; 96:907-914.