臺灣博碩士論文加值系統

泛過敏原 (Pan-allergen)具有高度保守序列和三維結構，會使不相關食物來源的過敏原產生IgE的交叉反應，導致過敏的病人產生嚴重的臨床症狀。Profilin、Lipid transfer proteins (LTP)和Pathogenesis-related protein 10 (PR-10)是植物來源食物中最重要的三種泛過敏原。檢測植物性食物裡泛過敏原濃度可以提供過敏病人正確的飲食建議，而台灣經常食用的植物來源食物之三種泛過敏原的濃度尚不清楚。本研究目的為量化台灣本地水果粗萃物裡三種泛過敏原。實驗方法首先由番茄的幼芽萃取mRNA，經由反轉錄聚合酶連鎖反應(RT-PCR)，產生profilin (rSola l 1)、nsLTP (rSola l 3)和 PR-10 (rSola l 4) 的cDNA。其次接合到pQE30載體並轉型在大腸桿菌的表現系統，最後進行表現及大量純化三種重組蛋白質。此外，我們製備18種本土或進口的水果粗萃物，並利用自製之免子anti-rSola l 1、3、4多株抗體，以immuno-dotted blotting方法分析三種泛過敏原的含量。實驗結果首先以純化的重組蛋白質rSola l 1、3、4進行ELISA，分析120位水果過敏的病人血清IgE反應，陽性率分別為rSola l 1, 13%；rSola l 3, 32%; rSola l 4, 13%。其次18種粗萃物泛過敏原定量結果顯示水蜜桃和芒果含有最高含量的profilin、nsLTP和PR-10；木瓜、香瓜和番茄也具有低含量的三種泛過敏原；而胡蘿蔔只有檢測到LTP的含量。本檢測最低靈敏濃度為0.65 μg/ml，結果顯示immuno-dotted blotting法足以檢測出植物性食物之低含量的泛過敏原。總結我們初步數據顯示，最常發生交叉反應的水果為水蜜桃和芒果，和西方文獻所描述的結果一致。我們成功建立了快速檢測食物泛過敏原的方法。

Background: Pan-allergens that share highly conserved sequences play a role in worsening the allergic symptoms of patients via IgE cross-reactions to allergens in unrelated sources. Measuring pan-allergen levels in plant food allow a correct dietary advice. Lipid transfer proteins (LTP), profilin and PR-10 are the most important pan-allergens among plant-derived food. However, concentrations of the three pan-allergens in local common consumed plant food are unknown.
Objective: This study aimed to characterize and quantify these pan-allergens in local fruit extracts.
Materials and methods: We isolated mRNA of Solanum lycopersicum and synthesized cDNA library by RT-PCR. cDNA encoding profilin (Sola l 1), LTP (Sola l 3) and PR-10 (Sola l 4) were cloned into the pQE30 and expressed in the Escherichia coli. In addition, we prepared crude extracts by rapid aqueous extraction with PBS from 18 kinds of local or imported plant foods. The extracts were analyzed for the pan-allergen levels by immunodot blotting using rabbit anti-rSola l 1, 3 and 4 polyclonal antibodies.
Results: The purified recombinant Sola l 1, Sola l 3 and Sola l 4 were recognized by 13%, 32% and 13% of 120 human sera tested by in-house ELISA, respectively. Among 18 tested plant extracts, peach and mango contained the highest levels of profilin, LTP and PR-10. Papaya, melon and tomato contained low but detectable levels of the three pan-allergens. Carrot only measured the level of LTP, but not profilin and PR-10. With a detection limit of 0.625 μg/ml, the sensitivity of the dot blotting should be sufficient to detect even low levels of pan-allergens in plant food products.
Conclusions: Our preliminary data revealed that the most involved fruit of cross-reactivity seem to be the peach and mango, which confirmed the literature described. We have successfully established a direct dot-blotting protocol for semi-quantification of plant-derived pan-allergens.

中文摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VI
表目錄 VII
縮寫對照表 (Abbreviation) VIII
第一章 前言 1
第一節. 食物過敏之流行病學和簡史 1
第二節. 食物過敏的類型 2
第三節. 食物過敏原 3
第四節. 植物性食物之泛過敏原 4
第五節. 食物過敏原的診斷和治療 6
第六節. 研究目的 7
第七節. 實驗設計 7

第二章 實驗材料與方法 8
第一節. 實驗材料 8
1. 植物材料 8
2. 實驗菌種與質體 8
第二節. 化學藥品、試劑 9
1.一般化學試劑：藥品大部分請購Merck與Sigma 9
2.限制酶及核酸修飾酵素 9
3. DNA電泳相關藥品 9
4. 蛋白質電泳相關藥品 9
5. 引子(Primer) 10
第三節. 器材和儀器 10
1.電泳設備 10
2.桌上型離心機(Eppendorf 5417) 10
3.其他器材 10
第四節. 實驗方法 10
1. 番茄過敏原profilin (Sola l 1)，nsLTP (Sola l 3)和 PR-10 (Sola l 4)之基因選殖 10
2. 重組蛋白質表現及純化 14
3. 蛋白質電泳分析 15
4. IgE ELISA 16
5. 多株忼體製備 17

第三章 實驗結果 18
第一節. 泛過敏原profilin (Sola l 1)，nsLTP (Sola l 3)和 PR-10 (Sola l 4)之基因選殖與序列比對分析 18
第二節. 重組蛋白質之表現 18
第三節. 重組蛋白質純化 19
第四節. 紐西蘭兔之多株抗體製備 19
第五節. 重組泛過敏原對水果過敏的病人血清之盛行率 19
第六節. Immuno-dotted blotting分析 20

第四章 討論 21
第一節. 泛過敏原Profilin、nsLTP和PR-10的分子特性及臨床關聯性 21
第二節. Immuno-dotted blotting的實際應用 21
第三節. 過敏原的臨床應用 22
第四節. 結論 22

第五章 參考文獻 23

附錄 41
附錄一. 三種重組過敏原引子序列與限制酶切位 41
附錄二. Three-dimensional structures of plant pan-allergens 42
附錄三. Alignments of amino acid sequences of tomato Sola l 1 43
附錄四. Alignments of amino acid sequences of tomato Sola l 3 44
附錄五. Alignments of amino acid sequences of tomato Sola l 4 45
附錄六. 兔子動物實驗流程 46
附錄七. pQE-30 Vectors 47
附錄八. pCR 2.1 48

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衛生福利部食品藥物管理署(2018)。公告食品過敏原標示規定，應標示之過敏原增加為11項。2018年08月21日。