Chula Vaccine Research Center, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Stallergenes-Greer, Antony, France.
Clin Exp Allergy. 2018 Apr;48(4):464-474. doi: 10.1111/cea.13098. Epub 2018 Feb 22.
Although the house dust mite species Blomia tropicalis is a leading cause of allergic diseases in tropical and subtropical regions, the identification and characterization of the allergenic proteins remain incomplete.
We aimed to characterize a recombinant form of Blo t 7 (rBlo t 7) in terms of IgE reactivity, lipid-binding activity and ability to stimulate innate immunity.
The mature Blo t 7 cDNA was cloned by PCR methods for the expression of a secreted form of the allergen in P. pastoris. The IgE reactivity to purified rBlo t 7 as well as the potential cross-reactivity with Der p 7 was determined by ELISA. The lipid-binding capacity of rBlo t 7 was assayed using fluorescent lipid probes. The stimulation of TLR2 signalling pathway by rBlo t 7 was examined in cell activation and reporter assays.
The amplified mature Blo t 7 cDNA revealed the presence of a 60 base pair insertion compared with the reference sequence registered in the GenBank database. Multiple protein sequence alignments of group 7 mite allergens confirmed that this longer deduced amino acid sequence was the authentic Blo t 7 polypeptide chain. Analysis of IgE reactivity can classify rBlo t 7 as an intermediate B. tropicalis allergen which displayed weak cross-reactivity with Der p 7. Purified rBlo t 7 was shown to bind selectively the naturally fluorescent lipid probe cis-parinaric (cPNA) with a dissociation constant of 2 μmol/L. The group 7 Blomia allergen stimulated the TLR2-, NF-kB- and MAPK-dependent production of IL-8 and GM-CSF in respiratory epithelial cells.
CONCLUSIONS & CLINICAL RELEVANCE: Through its propensity to transport fatty acids/lipids and to stimulate TLR2 signalling pathways in airway epithelial cells, Blo t 7 can represent a key allergen for the initiation of the B. tropicalis-induced airway inflammation.
尽管热带屋尘螨物种 Blomia tropicalis 是热带和亚热带地区过敏疾病的主要原因，但过敏原蛋白的鉴定和表征仍不完整。
我们旨在从 IgE 反应性、脂质结合活性和刺激固有免疫的能力方面对重组 Blo t 7（rBlo t 7）进行表征。
通过 PCR 方法克隆成熟的 Blo t 7 cDNA，用于在 P. pastoris 中表达过敏原的分泌形式。通过 ELISA 测定纯化的 rBlo t 7 的 IgE 反应性以及与 Der p 7 的潜在交叉反应性。使用荧光脂质探针测定 rBlo t 7 的脂质结合能力。通过细胞激活和报告基因测定检测 rBlo t 7 对 TLR2 信号通路的刺激作用。
与 GenBank 数据库中注册的参考序列相比，扩增的成熟 Blo t 7 cDNA 显示存在 60 个碱基对的插入。7 组螨过敏原的多个蛋白质序列比对证实，这种更长的推导氨基酸序列是真正的 Blo t 7 多肽链。IgE 反应性分析可将 rBlo t 7 归类为中间 B. tropicalis 过敏原，与 Der p 7 显示出较弱的交叉反应性。纯化的 rBlo t 7 被证明可以选择性地结合天然荧光脂质探针顺式-parinaric（cPNA），解离常数为 2 μmol/L。7 组 Blomia 过敏原刺激呼吸道上皮细胞中 TLR2、NF-kB 和 MAPK 依赖性 IL-8 和 GM-CSF 的产生。
通过其转运脂肪酸/脂质的倾向和刺激气道上皮细胞中的 TLR2 信号通路，Blo t 7 可以代表引发 B. tropicalis 诱导的气道炎症的关键过敏原。
