Ahrazem Oussama, Ibáñez M Dolores, López-Torrejón Gema, Sánchez-Monge Rosa, Sastre Joaquin, Lombardero Manuel, Barber Domingo, Salcedo Gabriel
Unidad de Bioquímica, Departamento de Biotecnología, ETS Ingenieros Agrónomos, UPM, Madrid, Spain.
Int Arch Allergy Immunol. 2005 Jul;137(3):201-10. doi: 10.1159/000086332. Epub 2005 Jun 9.
Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy.
Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3.
The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens.
Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30-50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.
脂质转移蛋白（LTPs）是重要的水果过敏原，最近被提议作为植物性食物过敏原模型。迄今为止，尚未对柑橘类水果过敏原进行鉴定。我们试图鉴定和分离柑橘类水果LTPs，并探讨它们与橙子过敏的相关性。
选取27例患者，这些患者在摄入橙子后主要表现为口腔过敏综合征，对橙子的点刺试验呈阳性且血清特异性IgE水平升高。通过色谱方法分离天然橙子和柠檬LTPs，以及在毕赤酵母中表达的重组橙子LTP异构体，通过N端氨基酸测序、基质辅助激光解吸/电离质谱对其进行鉴定，对于重组过敏原，对相应的cDNA进行DNA测序。用这三种纯化的过敏原以及主要的桃子LTP过敏原Pru p 3进行特异性IgE测定、免疫检测、ELISA抑制试验和体内皮肤点刺试验（SPT）。
从橙子（nCit s 3）和柠檬（nCit l 3）中纯化的天然过敏原显示出非常相似的N端氨基酸序列（20个残基中有18个相同），这是LTPs的典型特征，分子量分别为9610 Da和9618 Da。在毕赤酵母中表达的重组橙子异构体（rCit s 3）（N端区域20个残基中有16个与其天然对应物相同）有92个氨基酸残基，分子量为9463 Da，与rPru p 3的序列同一性为67%。在分析的27份血清中，54%的血清对纯化的nCit l 3过敏原呈特异性IgE阳性，48%对nCit s 3呈阳性，46%对rCit s 3呈阳性，37%对rPru p 3呈阳性。在26例检测nCit s 3的患者中，7例点刺试验呈阳性，8例检测nCit l 3的患者中3例呈阳性，26例检测nPru p 3的患者中10例呈阳性。ELISA抑制试验显示天然和重组橙子LTPs具有相同的IgE结合模式，纯化的橙子、柠檬和桃子LTP过敏原之间存在IgE交叉反应性。
LTP过敏原家族成员与橙子过敏有关，在30%-50%的研究患者中表现出体外和体内阳性反应。橙子和柠檬过敏原均与主要的桃子过敏原Pru p 3存在交叉反应性。
