乳膠-水果症候群之印度棗交叉性過敏原分子特性的研究

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Title:	乳膠-水果症候群之印度棗交叉性過敏原分子特性的研究
Other Titles:	Molecular characterization of Indian jujube (Zizyphus mauritiana)cross-reacting allergens in latex-fruit syndrome
Authors:	李美芳 Lee, Mey-Fann
Contributors:	黃光裕;吳啟輝 Hwang, Guang-Yuh;Wu, Chii-Huei 東海大學生命科學系
Keywords:	乳膠過敏症;交叉性反應;乳膠-水果症候群;過敏原 latex allergy;cross-reactivity;latex-fruit syndrome;allergen
Date:	2005
Issue Date:	2011-04-15T01:33:30Z (UTC)
Abstract:	天然乳膠是巴西橡膠樹(Hevea brasiliensis)樹幹流出的乳汁，多年來被廣泛地用來製造各種醫療器材。乳膠過敏症第一個病例報告在1979年由Nutter發表，近年來已經成為一個重要的國際性健康問題。已知乳膠過敏原是多種分子量分佈於10~100 kDa的蛋白質，目前至少有13種乳膠過敏原被選殖並命名為Hev b 1~Hev b 13。許多研究發現，有超過一半的乳膠過敏病人，對植物性食物尤其是水果，有專一性的IgE抗體。這種交叉性過敏反應，在1994年訂名為乳膠-水果症候群。目前參與此類交叉作用的共同抗原表位尚未被鑑定出來。不論引起過敏反應的重要性如何，在台灣還沒有關於乳膠過敏症和乳膠-水果症候群的深入研究。印度棗是一種美味的水果，普遍種植在南台灣。本研究有三個目的依序進行。第一是從印度棗和鹼化乳膠原料製備粗萃物，利用二位乳膠-印度棗過敏病人之血清，進行過敏原專一性的IgE抗體測定、免疫轉漬法、免疫轉漬抑制法和ELISA抑制法等實驗。我們鑑識一種42 kDa的乳膠過敏原和兩種分子量分別為30和42 kDa的印度棗過敏原，並且證實印度棗和乳膠過敏原之間，存在交叉性的抗原決定因子。胜?質量指紋鑑定的結果顯示，印度棗30 kDa過敏原和稻米(Oryza sativa)的Class III chitinase有同源性，而乳膠42 kDa過敏原則是Hev b 7。第二是從印度棗mRNA構築一個ZAP II cDNA基因庫，利用IgE抗體進行免疫篩選，共獲得10株呈陽性反應的溶菌斑。cDNA的長度在0.15~1.39 kb，並且進行核?酸定序。轉譯30 kDa過敏原的重組株訂名為Ziz m 1，利用酵母菌Pichia pastoris表現重組過敏原(rZiz m 1)。rZiz m 1擁有chitinase的活性，對8位乳膠-印度棗過敏病人，有87.5%的陽性皮膚反應。另外，由電腦的比對搜尋發現，Ziz m 1序列和hevamine (乳膠的class III chitinase)有45%的相似性。第三是從8種不同批號的乳膠手套製備乳膠粗萃物，共有20位對乳膠過敏的醫護人員參與研究。由免疫轉漬法發現3種IgE結合蛋白質，分子量分別為45、30和14 kDa。胜?質量指紋鑑定的結果，證明均為已知乳膠過敏原，依序是Hev b 13、hevamine和Hev b 1。我們的研究證明hevamine是一種重要的交叉性過敏原，和台灣醫護人員的乳膠-水果症候群有關，這個結果和西方國家的報導並不相同。將來我們將致力於Ziz m 1分子之IgE結合抗原表位的分析。對於過敏原的特性有充分瞭解，可以促進發展有效用的免疫治療，也可能解答乳膠-水果症候群的致病機轉。 Natural rubber latex is the milky sap obtained by tapping the bark of Hevea brasiliensis trees, and it is a common component in various medical equipments. Latex allergy was first described by Nutter in 1979 and has been recognized as a very important international health problem in the last few years. It has been demonstrated that latex allergens are various proteins with molecular weights ranging from 10 to 100 kilodaltons (kDa), and at least 13 latex allergens (Hev b 1~b 13) have been cloned and named. Several studies have shown that more than half of latex-sensitized people had specific IgE antibodies to proteins from many plant foods, especially fruits. This type of cross-reactivity is designated as the latex-fruit syndrome in 1994. However, the common epitopes responsible for these associations have not been well elucidated.Despite the importance in the elicitation of hypersensitivity reactions, the latex allergy and latex-fruit syndrome have not been closely studied in Taiwan. Indian jujube (Zizyphus mauritiana) is a sweet fruit that is abundantly cultivated in southern Taiwan. There were three aims of the present study and carried out sequentially. Firstly, the crude extracts were prepared from Indian jujube and ammoniated latex. Sera from two latex-Indian jujube allergic subjects were used for allergen-specific IgE, immunoblotting, immunoblot inhibition and ELISA inhibition studies. We identified a 42 kDa latex allergen and two allergens, 30 and 42 kDa, from Indian jujube and proved that the cross-reactive determinants exist between Indian jujube and latex allergens. Peptide mass fingerprinting revealed that the 30 kDa-Indian jujube allergen was homologous with class III chitinase of Oryza sativa and the 42-kDa latex protein was identified as a known allergen, Hev b 7.Secondly, a ZAP II cDNA library was constructed from Z. mauritiana mRNA and screened with IgE antibodies. Ten immunoreactive clones containing inserts ranging from 0.15 to 1.39 kb were identified and sequenced. The Ziz m 1 encoding 30 kDa allergen was expressed in Pichia pastoris. The recombinant Ziz m 1 (rZiz m 1) showed chitinase activity and possessed 87.5% skin response on eight latex-Indian jujube sensitive subjects. Moreover, the computer-assisted homology search revealed that Ziz m 1 with 45% sequence similarity to hevamine (class III chitinase of H. brasiliensis).Thirdly, crude extracts from eight different lots of latex gloves were prepared and 20 medical workers with latex allergy were included in the study. Immunoblotting revealed that three IgE-binding proteins, at molecular mass of 45, 30 and 14 kDa, were identified. Peptide mass fingerprinting demonstrated that they were known latex allergens Hev b 13, hevamine and Hev b 1, respectively.Our results indicated that hevamine is an important cross-reactive allergen associated with latex-fruit syndrome among medical workers in Taiwan, which is different from the reports in Western countries. Future works will focus on the analysis of immunodominant IgE-binding epitopes of the Ziz m 1 molecule. The well characterized allergen will aid in developing effective immunotherapy and may help to elucidate the mechanism of latex-fruit syndrome.
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