Department of Allergy, Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing Key Laboratory of Precision Medicine for Diagnosis and Treatment on Allergic Diseases, Beijing 100730, P.R. China.
Research Division of Clinical Pharmacology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
Int J Mol Med. 2019 Mar;43(3):1531-1541. doi: 10.3892/ijmm.2019.4065. Epub 2019 Jan 15.
Dogs are a major source of indoor allergens. However, the prevalence of dog allergies in China remains unclear, especially in children. In the present study, Can f 7, a canine allergen belonging to the Niemann pick type C2 protein family, was selected to study its sensitization rate in Chinese children with dog allergies. The Can f 7 gene was subcloned into a pET‑28a vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant Can f 7 was purified by nickel affinity chromatography, identified by SDS‑PAGE electrophoresis, and had its allergenicity assessed by western blot, ELISA and basophil activation tests. Through a series of bioinformatical approaches, B‑cell epitopes, secondary structures, and 3 dimensional (3D) homology modeling of Can f 7 were predicted. The activity of the B cell epitopes was verified by ELISA. The recombinant Can f 7 showed a distinct band with a molecular weight of 14 kDa. Six of 20 sera from dog‑allergic children reacted positively to the Can f 7. Can f 7 induced an ~4.0‑fold increase in cluster of differentiation 63 and C‑C motif chemokine receptor R3 expression in basophils sensitized with the serum of dog‑allergic children compared with those of non‑allergic controls. The secondary structure analysis showed that Can f 7 contains 6 β‑sheets. Five B cell epitopes of Can f 7 were predicted, and two of these were confirmed by ELISA. These results indicate that Can f 7 is an important canine allergen in Chinese children and provide novel data for further research concerning the use of Can f 7 in the diagnosis and treatment of Chinese children with canine allergy symptoms.
狗是室内过敏原的主要来源。然而，狗过敏在中国的流行情况尚不清楚，尤其是在儿童中。本研究选择犬过敏原 Can f 7（属于 Niemann pick 型 C2 蛋白家族），研究其在中国儿童狗过敏中的致敏率。将 Can f 7 基因亚克隆到 pET-28a 载体中，并在大肠杆菌 BL21 (DE3) 细胞中表达。通过镍亲和层析纯化重组 Can f 7，通过 SDS-PAGE 电泳鉴定，通过 Western blot、ELISA 和嗜碱性粒细胞激活试验评估其变应原性。通过一系列生物信息学方法预测 Can f 7 的 B 细胞表位、二级结构和三维（3D）同源建模。通过 ELISA 验证 B 细胞表位的活性。重组 Can f 7 显示出一条明显的带，分子量为 14 kDa。20 份来自狗过敏儿童的血清中有 6 份对 Can f 7 呈阳性反应。与非过敏对照相比，Can f 7 诱导狗过敏儿童血清致敏的嗜碱性粒细胞中 CD63 和 C-C 基序趋化因子受体 R3 的表达增加了约 4.0 倍。二级结构分析表明，Can f 7 含有 6 个β-折叠。预测了 Can f 7 的 5 个 B 细胞表位，其中 2 个通过 ELISA 得到证实。这些结果表明 Can f 7 是中国儿童中一种重要的犬过敏原，为进一步研究 Can f 7 在诊断和治疗中国儿童犬过敏症状中的应用提供了新的数据。
