Lin Jianli, Wang Hui, Li Meng, Liang Zhilin, Jiang Congli, Wu Yulan, Liu Zhigang, Yang Pingchang, Liu Xiaoyu
Institute of Allergy & Immunology, Shenzhen University Shenzhen 518060, China.
Am J Transl Res. 2016 Feb 15;8(2):568-77. eCollection 2016.
This study aims to acquire a recombinant allergen of Der f 29b by cloning and expression, and to identify its immunogenicity. In this study, the total RNA of D. farinae was extracted, cloned and expressed based on the Der f 29b gene. The molecular characteristics of Der f 29b was analyzed by the procedures of Bioinformatics. The allergenicity of recombinant Der f 29b protein was examined by western-blotting, ELISA, Immune inhibitory assays and skin prick test. The gene of Der f 29b consisted of 495 bases, derived from its nucleic acid sequence and encoded 164 amino acids. Positive responses to r-Der f 29b were shown in 24.3% by means of skin prick testing with 37 DM-allergic patients. The immunoblotting assays demonstrated that serum IgE from allergic patients reacted to r-Der f 29b protein. The IgE reactivity of r-Der f 29b in the serum from r-Der f 29b allergic patients was increased by more than 2 folds compared with healthy subjects. Immune inhibition assays showed that the IgE cross-reactivity was between r-Der f 29b and DME. Bioinformatics analysis predicted four peptides (13-17, 67-71, 104-109 and 147-155) as the B cell epitopes and five peptides (5-14, 16-31, 35-43, 52-63 and 87-97) as the T cell epitopes. Secondary structure prediction of Der f 29b with software PSIPRED identified two α-helices and seven β-sheets in Der f 29b. In conclusion, Derf 29b protein was identified as a novel subtype of dust mite allergen.
本研究旨在通过克隆和表达获得重组变应原Der f 29b，并鉴定其免疫原性。在本研究中，提取了粉尘螨的总RNA，基于Der f 29b基因进行克隆和表达。通过生物信息学程序分析Der f 29b的分子特征。采用蛋白质印迹法、酶联免疫吸附测定法、免疫抑制试验和皮肤点刺试验检测重组Der f 29b蛋白的变应原性。Der f 29b基因由495个碱基组成，从其核酸序列推导而来，编码164个氨基酸。对37名粉尘螨过敏患者进行皮肤点刺试验，结果显示24.3%的患者对重组Der f 29b呈阳性反应。免疫印迹分析表明，过敏患者的血清IgE与重组Der f 29b蛋白发生反应。与健康受试者相比，重组Der f 29b过敏患者血清中重组Der f 29b的IgE反应性增加了2倍以上。免疫抑制试验表明，重组Der f 29b与粉尘螨提取物之间存在IgE交叉反应性。生物信息学分析预测了四个肽段（13 - 17、67 - 71、104 - 109和147 - 155）为B细胞表位，五个肽段（5 - 14、16 - 31、35 - 43、52 - 63和87 - 97）为T细胞表位。使用PSIPRED软件对Der f 29b进行二级结构预测，确定Der f 29b中有两个α螺旋和七个β折叠。总之，Der f 29b蛋白被鉴定为一种新型尘螨变应原亚型。
