Preparation of IgE Antibody and Distribution of IgE+ Secretory Cells in the Palatine Tonsil of Bactrian Camel
Preparation of IgE Antibody and Distribution of IgE+ Secretory Cells in the Palatine Tonsil of Bactrian Camel
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DOI：
10.2174/0929866530666221212115739
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发表时间：
2023-01-01
影响因子：
1.6
通讯作者：
Wang, Wen-Hui
中科院分区：
文献类型：
--
作者：
Liu, Li-Ping;Li, Min;Wang, Wen-Hui
背景沙尘引起的过敏性疾病已严重威胁人类健康，而双峰驼可长期生活在桑迪环境中。目的制备兔抗双峰驼IgE抗体，探讨腭扁桃体中IgE（+）分泌细胞的分布特点，为双峰驼腭扁桃体局部抗体的分布及免疫功能研究奠定理论基础。方法比较双峰驼IgE、伊加、IgM和IgG重链恒定区的氨基酸序列，筛选特异性IgE基因片段（447 bp）。将重组质粒pET-28 a-IgE转化大肠杆菌BL 21（DE 3），经IPTG诱导表达，并优化表达条件。用纯化的IgE重组蛋白免疫家兔制备抗体，用间接ELISA和Western blotting检测抗体的效价和特异性。免疫组织化学和统计学方法研究了腭扁桃体中IgE（+）分泌细胞的分布。结果重组蛋白以包涵体形式表达，大小约为16 kDa。最佳诱导浓度为0.7mmol/L，诱导时间为8h。ELISA检测抗体效价为1：16000，Western blotting检测抗体能与重组蛋白特异性结合。在双峰驼腭扁桃体中，IgE（+）分泌细胞主要分布于网状隐窝上皮的上皮下，其次为复层鳞状上皮的上皮下，偶见滤泡外区。结论成功制备了兔抗双峰驼IgE多克隆抗体。证实正常生活条件下双峰驼腭扁桃体中存在IgE。此外，IgE（+）分泌细胞主要分布于腭扁桃体网状隐窝上皮的上皮下，这与双峰驼腭扁桃体IgG（+）和sIgA（+）分泌细胞的分布特征一致。
Background Allergic diseases induced by dust have seriously threatened human health, while Bactrian camels can live in a sandy environment for a long time. Objective To prepare rabbit anti-Bactrian camel IgE antibody and explore the distribution characteristics of IgE(+) secretory cells in the palatine tonsils, which lays a theoretical foundation for the distribution of local antibodies in the palatal tonsils of Bactrian camel and the study of immune function. Methods In this study, the amino acid sequences of Bactrian camel IgE, IgA, IgM and IgG heavy chain constant regions were compared, and a specific IgE gene fragment were selected (447 bp). The recombinant plasmid pET-28a-IgE was induced in Escherichia coli BL21(DE3) by IPTG and its expression conditions were optimized. The antibody was prepared by immunizing rabbits with purified IgE recombinant protein, its titer and specificity were detected by indirect ELISA and Western blotting. Immunohistochemical and statistical methods investigated the distribution of IgE(+) secretory cells in the palatine tonsils. Results The IgE recombinant protein was expressed in the form of inclusion bodies with a size of 16 kDa. The optimal IPTG induction concentration was 0.7 mmol/L and the induction time was 8 h. The titer of the antibody was 1:16000 by ELISA, and the antibody could specifically bind to the recombinant protein by Western blotting. IgE(+) secretory cells were mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil of the Bactrian camel, followed by the subepithelial compartments of stratified squamous epithelium and occasionally in the extrafollicular region. Conclusion The rabbit anti-Bactrian camel IgE polyclonal antibody was successfully prepared. It is confirmed that IgE exists in the palatine tonsils of Bactrian camels under normal living conditions. In addition, IgE(+) secretory cells are mainly distributed in the subepithelial compartments of reticulated crypt epithelium of the palatine tonsil, which is consistent with the distribution characteristics of IgG(+) and sIgA(+) secretory cells in the palatal tonsils of the Bactrian camel.