Cai Ze-Lang, Liu Shan, Li Wei-Yong, Zhou Zi-Wen, Hu Wan-Zhen, Chen Jia-Jie, Ji Kunmei
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shenzhen University Medical School, Shenzhen 518060, China.
Shenzhen University General Hospital, Shenzhen 518060, China.
World Allergy Organ J. 2023 Aug 1;16(8):100804. doi: 10.1016/j.waojou.2023.100804. eCollection 2023 Aug.
House dust mites (HDMs), including (Der p) and (Der f) species, represent a major source of inhalant allergens that induce IgE-mediated anaphylactic reactions. HDM allergen identification is important to the diagnosis and treatment of allergic diseases. Here, we report the identification of a novel HDM allergen, which we suggest naming Der f 40, and its immunodominant IgE epitopes.
The recombinant protein Der f 40 was expressed using a pET prokaryotic expression system and purified with Ni-NTA resins. IgE binding activity was evaluated by IgE-western blot, dot-blot, and ELISA. Mast cell activation testing was performed to assess the cellular effects of IgE binding in mouse bone marrow derived mast cells (BMMCs) expressing human FcεRI. IgE binding assays were performed with truncated and hybrid Der f 40 protein molecules to find immunodominant IgE epitopes.
A 106-amino acid (aa) recombinant Der f Group 40 protein (rDer f 40) was obtained (GenBank accession No. XP_046915420.1) as thiredoxin-like protein. Der f 40 was shown to bind IgE from HDM allergic serum (9.68%; 12/124 in IgE-ELISA), and shown to promote the release of β-hexosaminidase from BMMCs dose-dependently when administered with HDM allergic sera. The Der f Group 40 protein was named Der f 40 and listed in the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. IgE binding assays with Der f 40-based truncated and hybrid proteins indicated that IgE binding epitopes are likely located in the C-terminal region and dependent on conformational structure. The 76-106-aa region of C-terminus was identified as an immunodominant IgE epitope of Der f 40.
A novel HDM allergen with robust IgE binding activity was identified and named Der f 40. An immunodominant IgE epitope of Der f 40 with conformational dependency was identified in the C-terminus (aa 76-106). These findings provide new information that may be useful in the development of diagnostic and therapeutic agents for HDM allergy.
屋尘螨（HDMs），包括狄斯瓦螨（Der p）和户尘螨（Der f）种类，是吸入性过敏原的主要来源，可引发免疫球蛋白E（IgE）介导的过敏反应。HDM过敏原鉴定对过敏性疾病的诊断和治疗很重要。在此，我们报告一种新型HDM过敏原的鉴定，我们建议将其命名为Der f 40，以及其免疫显性IgE表位。
使用pET原核表达系统表达重组蛋白Der f 40，并用镍-亚氨基三乙酸（Ni-NTA）树脂进行纯化。通过IgE-蛋白质免疫印迹法、斑点印迹法和酶联免疫吸附测定（ELISA）评估IgE结合活性。进行肥大细胞活化试验，以评估IgE结合对表达人高亲和力IgE受体（FcεRI）的小鼠骨髓来源肥大细胞（BMMCs）的细胞效应。用截短的和杂交的Der f 40蛋白分子进行IgE结合试验，以寻找免疫显性IgE表位。
获得了一种106个氨基酸（aa）的重组Der f第40组蛋白（rDer f 40）（GenBank登录号：XP_046915420.1），为类硫氧还蛋白。Der f 40被证明可结合HDM过敏血清中的IgE（在IgE-ELISA中为9.68%；124例中有12例），并且当与HDM过敏血清一起给药时，可剂量依赖性地促进BMMCs释放β-己糖胺酶。Der f第40组蛋白被命名为Der f 40，并列入世界卫生组织和国际免疫学会联盟（WHO/IUIS）过敏原命名小组委员会。用基于Der f 40的截短和杂交蛋白进行的IgE结合试验表明，IgE结合表位可能位于C末端区域，且依赖于构象结构。C末端的76-106-aa区域被确定为Der f 40的免疫显性IgE表位。
鉴定出一种具有强大IgE结合活性的新型HDM过敏原，并将其命名为Der f 40。在C末端（氨基酸76-106）鉴定出Der f 40的一个具有构象依赖性的免疫显性IgE表位。这些发现提供了可能有助于开发HDM过敏诊断和治疗药物的新信息。
