Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295, USA.
Int Arch Allergy Immunol. 2011;156(3):267-81. doi: 10.1159/000323887. Epub 2011 Jun 29.
IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity.
Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot.
IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50%) and 5 of 18 patients (28%), respectively. Four patients (22%) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera.
rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.
已在杏仁中鉴定出 IgE 反应性蛋白；然而，仅有少数已被克隆并针对特定患者 IgE 反应性进行了测试。在此，我们克隆并表达了主要杏仁蛋白普鲁宁的同工型普鲁宁 1 和普鲁宁 2，一种 11S 球蛋白，并对每种蛋白进行了 IgE 反应性检测。
从杏仁 cDNA 文库中通过 PCR 扩增普鲁宁同工型，测序，在大肠杆菌中克隆并表达。通过斑点印迹和免疫印迹分析，使用杏仁过敏患者的血清和鼠单克隆抗体（mAb）筛选重组（r）过敏原 Pru du 6.01 和 Pru du 6.02 的反应性。通过固相重叠肽分析鉴定连续 IgE 结合表位。通过免疫印迹分析评估变性重组蛋白的表位稳定性。
在 18 名患者中的 9 名（50%）和 18 名患者中的 5 名（28%）中发现了对 rPru du 6.01 和 rPru du 6.02 的 IgE 反应性。有 4 名患者（22%）对两种同工型均有反应性。鼠抗杏仁 IgG mAb 对 rPru du 6.01 的反应性也强于 rPru du 6.02。检测到稳定和不稳定的表位。使用 pooled 杏仁过敏患者血清，在 Pru du 6.01 上检测到 6 个具有 IgE 结合顺序表位的肽段，在 Pru du 6.02 上检测到 8 个。
在我们的患者群体中，rPru du 6.01 比 rPru du 6.02 更广泛地被识别。鉴定出多个连续表位，并观察到在一些患者中，用变性剂处理对 IgE 结合强度几乎没有影响，这表明普鲁宁上的连续表位起着重要作用。
