Analysis of IgE antibodies from a patient with atopic dermatitis: Biased V gene usage and evidence for polyreactive IgE heavy chain complementarity-determining region 3
Analysis of IgE antibodies from a patient with atopic dermatitis: Biased V gene usage and evidence for polyreactive IgE heavy chain complementarity-determining region 3
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DOI：
10.4049/jimmunol.168.12.6305
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发表时间：
2002-06-15
影响因子：
4.4
通讯作者：
Collins, AM
中科院分区：
文献类型：
--
作者：
Edwards, MR;Brouwer, W;Collins, AM
为了更好地了解过敏反应中 V 基因的使用、特异性和 IgE 抗体的克隆起源，我们利用成年特应性皮炎患者的 mRNA 构建了组合抗体库。随机克隆的序列分析显示，33% 的克隆使用 IGHV6-1 H 链 V 基因片段，这是 V(H)6 基因家族的唯一成员。 IGHV6-1 很少用于表达的成人曲目；然而，它与胎儿来源的抗体有关。 V(H)6重排的特点包括互补决定区3短、IGHD7-27 D基因的频繁使用以及D-J连接处添加少量核苷酸。与 V(H)1、V(H)3 和 V(H)4 重排相比，突变水平也较低。该文库表达为噬菌体-Fab 融合体，并通过在鸡蛋过敏原卵类粘蛋白上淘选来选择特定的噬菌体。在表达为可溶性 IgE Fab 后，12 个克隆通过 ELISA 证明与卵类粘蛋白、脱脂奶和 BSA 结合。核苷酸测序证明 IGHV6-1 V 基因片段编码 12 个多重反应性 IgE Fab 中的每一个。根据这些克隆中的几个克隆的互补决定区 3 设计了环肽。环肽可结合自身和非自身抗原，包括卵类粘蛋白、人 IgG、破伤风类毒素以及人和牛冯维勒布兰德因子。这些结果表明，一些 IgE Ab 可能结合不止一种 Ag，这对于了解特应性皮炎等疾病中的多种敏感性具有重要意义。
To better understand V gene usage, specificity, and clonal origins of IgE Abs in allergic reactions, we have constructed a combinatorial Ab library from the mRNA of an adult patient with atopic dermatitis. Sequence analysis of random clones revealed that 33% of clones used the IGHV6-1 H chain V gene segment, the only member of the V(H)6 gene family. IGHV6-1 is rarely used in the expressed adult repertoire; however, it is associated with fetal derived Abs. Features of the V(H)6 rearrangements included short complementarity-determining region 3, frequent use of IGHD7-27 D gene, and little nucleotide addition at the D-J junction. There was also a low level of mutation compared with V(H)1, V(H)3, and V(H)4 rearrangements. The library was expressed as phage-Fab fusions, and specific phage selected by panning on the egg allergen ovomucoid. Upon expression as soluble IgE Fabs, 12 clones demonstrated binding to ovomucoid, skim milk, and BSA by ELISA. Nucleotide sequencing demonstrated that the IGHV6-1 V gene segment encoded each of the 12 multiply reactive IgE Fabs. A cyclic peptide was designed from the complementarity-determining region 3 of several of these clones. The cyclic peptide bound both self and nonself Ags, including ovomucoid, human IgG, tetanus toxoid, and human and bovine von Willebrand factor. These results suggest that some IgE Abs may bind more than one Ag, which would have important implications for understanding the multiple sensitivities seen in conditions such as atopic dermatitis.