建立番茄過敏原 Sola l 1 分析平台及評估數種番茄品種中 Sola l 1 含量

Abstract

番茄 (Solanum lycopersicum)，又名為西紅柿，為茄科茄屬番茄亞屬的多年生草本植物。在台灣，番茄是易誘發過敏的蔬果之一。番茄中的主要過敏原 Sola l 1 為一種肌動蛋白結合蛋白 (profilin)。這類的蛋白在高等植物物種中序列保守且結構相近，因此時常造成蔬果與花粉之間的交叉反應，誘導過敏發生。本研究的目的即是希望能藉由研究番茄中的過敏原，建立蔬果食品過敏原的分析平台和技術。研究分成四部份，第一部份試驗欲取得番茄過敏原之基因並生產重組蛋白，自牛番茄 ‘紅慧’ 中選殖 Sola l 1 基因，片段大小約 396 bp，將此片段進行構築並轉形至 E.coli 系統中生產 Sola l 1重組蛋白，利用快速蛋白質液相層析系統 (fast protein liquid chromatography, FPLC) 純化含 His-tag 的重組蛋白 rHis-Sola l 1，分子量約 34 kDa。第二部份以此蛋白作為抗原生產抗體，建立過敏原蛋白質的分析平台。特異性分析發現rSola l 1 之單株抗體專一性表現不佳，仍須進一步針對單株抗體的特異性予以篩選。第三部份以即時聚合酶鏈鎖反應 (real-time quantitative PCR, qPCR) 作為分析方法，建立核酸定量的分析平台。依番茄 profilin 過敏原 Sola l 1 序列上與其他番茄中 profilin 蛋白具有特異性的位置設計引子，最終選擇 real 5引子作為 Sola l 1特異性引子；看家基因引子則選擇 Actin 3 和 18 S 1 作為相對定量的引子。第四部份以建立之分析平台分析不同品種與成熟度的番茄中 Sola l 1 含量。以核酸平台分析，結果顯示番茄品種間 Sola l 1 mRNA 表現量有顯著差異；而以蛋白分析平台搭配生物影像處理分析軟體分析，結果顯示 Sola l 1 蛋白含量於不同品種間有所差異，但與核酸定量結果之趨勢不完全相同。另外，以 Tanya 番茄探討成熟度與過敏原含量相關性，分析番茄五個生長階段 Sola l 1 過敏原含量，結果顯示 Sola l 1 核酸表現量隨番茄果實成熟而逐漸下降，在果實過熟階段則大幅增加；蛋白分析則無此變化趨勢。以蛋白分析平台作為番茄過敏原 Sola l 1 的定量分析平台仍需再進行特異性評估。本研究建立之番茄過敏原核酸分析平台可定量 Sola l 1 表現，並證實不同番茄品種以及不同成熟度皆會影響過敏原表現。

Tomato (Solanum lycopersicum) is a perennial plant belonging to the nightshade family (Solanaceae). In Taiwan, tomato is one of the most frequent causes of allergy among vegetables. Sola l 1, a profiling is a major allergen in Tomato. Due to the high identity in amino acid sequence among plant profilins, cross reaction usually happens among tomato, other allergenic foods, and pollens. The aim of this study was to construct analytical platforms by using both immunochemical and molecular biological methods for tomato allergen Sola l 1. First of all, we cloned a 396 bp cDNA of Sola l 1 from tomato ’紅慧’ and produced recombinant protein with E. coli expression system. The recombinant protein was named rHis-Sola l 1, and its molecular weight was 34 kDa. Second, this protein was used as an antigen for mouse immunization and monoclonal antibodies production. However, the specificity of the monoclonal antibody against rHis-Sola l 1 protein was not as well as expected. The hybridoma for monoclonal antibody production had to be further selected. In the determination of Sola l 1 mRNA expression, the primer pairs of real 5, Actin3, and 18S1 yielded the optimal results in PCR efficiency and PCR quantitation slope for relative quantification. After the analytical platforms were constructed, we selected twenty tomato cultivars for determining the mRNA expression of Sola l 1. Furthermore, five growth stages of tomato ’Tanya’ were selected for discussing the relationship between growth stages and mRNA expression of Sola l 1. The results indicated that the mRNA expression of Sola l 1 over cultivars would be different, but the variation trend of the protein expression of Sola l 1 was not the same as mRNA expression. The mRNA expression of Sola l 1 would decrease along with early growth stages, but increased in the overipen stage of tomato’Tanya’; however, the variation trend in protein level was not significant. The quantitative analysis platform of Sola l 1 protein has to be further evaluated. Finally, we have constructed analytical platforms for evaluating tomato Sola l 1 allergens in gene level.