Zhou Dongmei, Ren Yaning, Zhou Ying, Liao Yuanfen, Cheng Qi, Chen Jinni, Yuan Cunyin, Zeng Dan, Cui Yubao
Clinical Research Center, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi, 214023, People's Republic of China.
Department of Pediatrics Laboratory, The Affiliated Children´s Hospital of Jiangnan University, Wuxi, 214023, People's Republic of China.
Int J Nanomedicine. 2025 Jul 30;20:9505-9516. doi: 10.2147/IJN.S527210. eCollection 2025.
We aimed to develop recombinant allergens on the nanoscale by using self-assembly property of ferritin and to investigate their immune response and protective effect.
The cDNA encoding Ftn-Blo t 2 was synthesized and cloned into pET-21a (+) vector, then expressed in BL21 (DE3). Recombinant proteins (rBlo t 2, rFtn, and rFtn-Blo t 2) were purified and their immunoreactivity confirmed for immunoreactivity by IgE-ELISA and Western blot. In vitro, the immunogenicity of rFtn-Blo t 2 was assessed using BEAS-2B human bronchial epithelial cells by measuring epithelial cytokine responses. In vivo, a murine airway inflammation model was established via sensitization and challenge with crude extract, followed by treatment with rFtn-Blo t 2 to investigate its effects on airway inflammation, Th1/Th2 cytokine profiles, and allergen-specific antibody production.
The average diameter of rFtn and rFtn-Blo t 2 particles are (11.24 ± 1.31) nm and (16.00 ± 1.59) nm. The IgE- binding rates of rFtn-Blo t 2 and rBlo t 2 were 62.5% (15/24) and 58.3% (12/24), respectively. The expressions of IL-25, IL-33, and TSLP increased by 3.29, 3.43, and 2.22 folds after the addition of rFtn-Blo t 2 in co-culture with BEAS-2B. Mice challenged with rBlo t 2 had lower levels of cytokines IL-13 and IL-4 but higher levels of IFN-γ and TGF-β in alveolar lavage fluid compared to controls (p < 0.05). HE staining showed that compared with rBlo t 2 group, the inflammation level in lung tissue of rFtn-Blo t 2 group was reduced.
A nano-scale allergen was successfully developed, providing a concept, a platform, and a paradigm for the construction of nano-scale allergens.
我们旨在利用铁蛋白的自组装特性开发纳米级重组变应原，并研究其免疫反应和保护作用。
合成编码Ftn-Blo t 2的cDNA并克隆到pET-21a(+)载体中，然后在BL21(DE3)中表达。纯化重组蛋白（rBlo t 2、rFtn和rFtn-Blo t 2），并通过IgE-ELISA和蛋白质印迹法确认其免疫反应性。在体外，通过测量上皮细胞因子反应，使用BEAS-2B人支气管上皮细胞评估rFtn-Blo t 2的免疫原性。在体内，通过用粗提物致敏和激发建立小鼠气道炎症模型，然后用rFtn-Blo t 2治疗，以研究其对气道炎症、Th1/Th2细胞因子谱和变应原特异性抗体产生的影响。
rFtn和rFtn-Blo t 2颗粒的平均直径分别为(11.24±1.31)nm和(16.00±1.59)nm。rFtn-Blo t 2和rBlo t 2的IgE结合率分别为62.5%（15/24）和58.3%（12/24）。在与BEAS-2B共培养中加入rFtn-Blo t 2后，IL-25、IL-33和TSLP的表达分别增加了3.29、3.43和2.22倍。与对照组相比，用rBlo t 2激发的小鼠肺泡灌洗液中细胞因子IL-13和IL-4水平较低，但IFN-γ和TGF-β水平较高（p<0.05）。苏木精-伊红染色显示，与rBlo t 2组相比，rFtn-Blo t 2组肺组织炎症水平降低。
成功开发了一种纳米级变应原，为纳米级变应原的构建提供了一个概念、一个平台和一个范例。
