Cloning, Expression and Patient IgE Reactivity of Recombinant Pru du 6, an 11S Globulin from Almond
Cloning, Expression and Patient IgE Reactivity of Recombinant Pru du 6, an 11S Globulin from Almond
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重组 Pru du 6（一种来自杏仁的 11S 球蛋白）的克隆、表达和患者 IgE 反应性
DOI：
10.1159/000323887
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发表时间：
2011
影响因子：
2.8
通讯作者：
K. Roux
中科院分区：
文献类型：
--
作者：
Leanna N Willison;Pallavi Tripathi;G. Sharma;S. Teuber;S. Sathe;K. Roux
背景：IgE反应蛋白已被确定在杏仁，但是，很少有克隆和测试特异性患者IgE反应。在这里，我们克隆和表达的主要杏仁蛋白李树素，11S球蛋白，李树素1和李树素2，同种型，并测定每个IgE反应性。方法：从扁桃cDNA文库中扩增Prunin异构体，测序、克隆并在大肠杆菌中表达。使用杏仁过敏患者血清和鼠单克隆抗体（mAb），通过斑点印迹和免疫印迹试验筛选对重组（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确定顺序IgE结合表位。通过免疫印迹分析变性重组蛋白来评估表位稳定性。结果：18例患者中分别有9例（50%）和5例（28%）对rPru du 6.01和rPru du 6.02有IgE反应。4例患者（22%）表现出对两种亚型的反应性。鼠抗杏仁IgG mAb对rPru du 6.01的反应性也高于对rPru du 6.02的反应性。稳定和不稳定表位均被检测到。使用合并的杏仁过敏血清检测到Pru du 6.01和Pru du 6.02上的6个IgE结合连续表位承载肽段。结论：在我们的患者人群中，rPru du 6.01比rPru du 6.02得到更广泛的认可。多个连续表位的鉴定和观察，与变性剂的治疗对IgE结合强度在一些患者中的影响不大，表明连续表位的重要作用。
Background: 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. Methods: 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. Results: 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. Conclusions: 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.
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DOI：
10.1016/j.str.2009.08.002
发表时间：
2009-09-09
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Kim MW;Chelliah Y;Kim SW;Otwinowski Z;Bezprozvanny I
通讯作者：
Bezprozvanny I
影响因子：
14.2
作者：
Teuber, SS;Dandekar, AM;Sellers, CL
通讯作者：
Sellers, CL
影响因子：
14.2
作者：
Sicherer, Scott H.;Munoz-Furlong, Anne;Sampson, Hugh A.
通讯作者：
Sampson, Hugh A.
影响因子：
15.9
作者：
Rabjohn, P;Helm, EM;Bannon, GA
通讯作者：
Bannon, GA
影响因子：
14.2
作者：
Sicherer, SH;Muñoz-Furlong, A;Sampson, HA
通讯作者：
Sampson, HA