Tinghino R, Barletta B, Palumbo S, Afferni C, Iacovacci P, Mari A, Di Felice G, Pini C
Department of Immunology, Istituto Superiore di Sanità, Rome, Italy.
J Allergy Clin Immunol. 1998 Jun;101(6 Pt 1):772-7. doi: 10.1016/S0091-6749(98)70306-9.
Species belonging to the Cupressaceae family are a relevant source of allergens that are present in a wide number of countries.
We sought to identify, purify, and characterize recombinant allergens from Juniperus oxycedrus, a species belonging to the Cupressaceae family.
Double-stranded cDNA was synthesized from mRNA and cloned into the lambda-ZAP expression vector. IgE screening of the library was performed with a pool of sera from subjects allergic to Cupressaceae. A recombinant 6xHis-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni2+ affinity chromatography. It was studied further by immunoblotting inhibition with pollen extracts from other Cupressaceae, Oleaceae, Urticaceae, and Graminaceae. The role of protein-bound calcium on the allergen's IgE-binding capacity was tested in a plaque assay in the presence or absence of EGTA.
A cDNA coding for a newly identified Juniperus oxycedrus pollen allergen, rJun o 2, was isolated. The deduced amino acid sequence contained four typical Ca2+ binding sites and showed a significant sequence similarity to calmodulins. Depletion of Ca2+ in the plaque assay led to a loss of IgE-binding capacity of rJun o 2. Immunoblotting inhibition revealed that J. oxycedrus, J. ashei, Cupressus arizonica, C. sempervirens, Parietaria judaica, Olea europaea, and Lolium perenne pollen extracts were able to inhibit IgE binding to blotted rJun o 2 at different concentrations.
rJun o 2 contains IgE-binding epitopes shared by taxonomically unrelated species, and therefore it can be regarded as a new panallergen. These findings could contribute to an explanation for the phenomenon of multiple positive test results in polysensitized patients and the potential symptom-eliciting role of allergenic sources previously not encountered.
柏科植物是广泛存在于许多国家的过敏原的重要来源。
我们试图从柏科植物刺柏中鉴定、纯化和表征重组过敏原。
从mRNA合成双链cDNA，并克隆到λ-ZAP表达载体中。用对柏科植物过敏的受试者的血清池对文库进行IgE筛选。一种重组的带有6xHis标签的刺柏过敏原Jun o 2在大肠杆菌中表达，并通过Ni2+亲和层析纯化。通过用其他柏科、木犀科、荨麻科和禾本科的花粉提取物进行免疫印迹抑制进一步研究。在有或没有乙二醇双四乙酸（EGTA）的情况下，在噬斑试验中测试蛋白质结合钙对过敏原IgE结合能力的作用。
分离出编码一种新鉴定的刺柏花粉过敏原rJun o 2的cDNA。推导的氨基酸序列包含四个典型的Ca2+结合位点，并且与钙调蛋白显示出显著的序列相似性。噬斑试验中Ca2+的耗尽导致rJun o 2的IgE结合能力丧失。免疫印迹抑制显示，刺柏、阿氏刺柏、亚利桑那柏、意大利柏、墙草、油橄榄和黑麦草的花粉提取物能够在不同浓度下抑制IgE与印迹的rJun o 2的结合。
rJun o 2含有分类学上不相关物种共有的IgE结合表位，因此可被视为一种新的泛过敏原。这些发现有助于解释多敏患者多次阳性检测结果的现象以及以前未遇到的过敏原来源的潜在症状引发作用。
