Reese G, Schicktanz S, Lauer I, Randow S, Lüttkopf D, Vogel L, Lehrer S B, Vieths S
Paul-Ehrlich-Institut, Department of Allergology, Langen, Germany.
Clin Exp Allergy. 2006 Apr;36(4):517-24. doi: 10.1111/j.1365-2222.2006.02454.x.
Recombinant allergens are considered the basis for new diagnostic approaches and development of novel strategies of allergen-specific immunotherapy. As Pen a 1 from brown shrimp Penaeus aztecus is the only major allergen of shrimp and binds up to 75% of all shrimp-specific IgE antibodies this molecule may be an excellent model for the usage of allergens with reduced IgE antibody-binding capacity for specific immunotherapy.
The aim was to clone, express and characterize a full-length recombinant Pen a 1 molecule and compare it with natural Pen a 1 in regard to structural and immunological parameters such as IgE antibody capacity and ability to induce IgE-mediated mediator release.
Total RNA was isolated from P. aztecus and a rapid amplification of cDNA ends (5' RACE) was performed to obtain full-length cDNA coding for Pen a 1. Using a gene-specific primer, PCR was performed and full-length cDNA was cloned and sequenced. Recombinant His-tagged Pen a 1 was isolated from Escherichia coli under native conditions by immobilized metal affinity chromatography. Secondary structure of natural and recombinant Pen a 1 was compared by circular dichroism (CD) spectroscopy, and the IgE antibody-binding capacity evaluated by RAST. The allergenic potency was tested by the capability of natural and recombinant Pen a 1 to induce mediator release in a murine and human in vitro model of IgE-mediated type I allergy.
The deduced amino-acid sequence was 284 residues long and amino-acid sequence identities with allergenic and non-allergenic tropomyosins ranged from 80% to 99% and 51% to 58%, respectively. The analysis of the secondary structure of natural and recombinant Pen a 1 by CD spectroscopic analysis showed that both nPen a 1 and rPen a 1 had alpha-helical conformation that is typical for tropomyosin. The IgE antibody binding capacities of nPen a 1 and r Pen a1 were found to be essentially identical by RAST. The mediator release experiments using both wild-type and humanized rat basophilic leukaemia 30/25 cells showed that rPen a 1 and nPen a 1 induced a similar level of mast cell activation.
Recombinant Pen a 1 and natural Pen a 1 are structurally and immunologically identical and rPen a 1 may be used as the basis for component-resolved diagnosis and the generation of modified shrimp tropomyosin for allergen-specific immunotherapy. The results of the animal studies indicate that C3H/HeJ mice that were sensitized with shrimp extract in combination with cholera toxin as adjuvant may be a suitable model to study shrimp allergy.
重组变应原被认为是新诊断方法和变应原特异性免疫治疗新策略开发的基础。由于来自墨西哥棕虾的Pen a 1是虾的唯一主要变应原，可结合高达75%的所有虾特异性IgE抗体，该分子可能是用于变应原特异性免疫治疗中使用IgE抗体结合能力降低的变应原的极佳模型。
目的是克隆、表达和表征全长重组Pen a 1分子，并在结构和免疫学参数（如IgE抗体结合能力和诱导IgE介导的介质释放的能力）方面将其与天然Pen a 1进行比较。
从墨西哥棕虾中分离总RNA，并进行cDNA末端快速扩增（5' RACE）以获得编码Pen a 1的全长cDNA。使用基因特异性引物进行PCR，克隆并测序全长cDNA。通过固定化金属亲和色谱在天然条件下从大肠杆菌中分离重组His标签的Pen a 1。通过圆二色性（CD）光谱比较天然和重组Pen a 1的二级结构，并通过RAST评估IgE抗体结合能力。通过天然和重组Pen a 1在IgE介导的I型过敏的小鼠和人类体外模型中诱导介质释放的能力来测试变应原效力。
推导的氨基酸序列长284个残基，与变应性和非变应性原肌球蛋白的氨基酸序列同一性分别为80%至99%和51%至58%。通过CD光谱分析对天然和重组Pen a 1的二级结构进行分析表明，nPen a 1和rPen a 1均具有原肌球蛋白典型的α-螺旋构象。通过RAST发现nPen a 1和rPen a 1的IgE抗体结合能力基本相同。使用野生型和人源化大鼠嗜碱性白血病30/25细胞进行的介质释放实验表明，rPen a 1和nPen a 1诱导的肥大细胞活化水平相似。
重组Pen a 1和天然Pen a 1在结构和免疫学上相同，rPen a 1可作为组分解析诊断的基础以及用于变应原特异性免疫治疗的修饰虾原肌球蛋白的生成基础。动物研究结果表明，用虾提取物与霍乱毒素作为佐剂致敏的C3H/HeJ小鼠可能是研究虾过敏的合适模型。
