人IgE Fc原核表达载体的构建、表达、纯化及对肥大细胞脱颗粒的抑制作用

Construction, Expression, Purification and Inhibition of Mast Cell Degranulation of Human IgE Fc Prokaryotic Expression Vector

目的探讨人IgE Fc原核表达载体的构建、表达、纯化及对肥大细胞脱颗粒抑制作用。方法以pEGFP-N1/IgE Fc真核表达载体为模版,用聚合酶链反应(PCR)方法扩增IgE Fc编码序列,通过镍柱亲和层析进一步纯化获得的蛋白质,采取酶联免疫吸附试验检测致敏肥大细胞中β-己糖胺酶和细胞因子。结果致敏肥大细胞中,抗原蛋白的表现形式主要是以涵体的模式存在且被成功纯化;pET-32a(+)-IgE Fc作用于未被致敏的RBL-2H3细胞后,RBL-2H3细胞的细胞膜平滑,细胞紧密贴覆于细胞壁,形态为梭形。结论pET-32 a(+)-IgE Fc可抑制肥大细胞的脱颗粒,但不会对致敏肥大细胞活性造成影响,为人IgE Fc和哮喘等过敏性疾病研究提供了理论基础。

Objective To discuss the construction,expression,purification and inhibition of mast cell degranulation of human IgE Fc prokaryotic expression vector.Methods Using pEGFP-N1/IgE Fc eukaryotic expression vector as template,the IgE Fc coding sequence was amplified by polymerase chain reaction(PCR).The protein was further purified by nickel column affinity chromatography.The beta-hexosaminase and cytokines in sensitized mast cells were detected by enzyme linked immunosorbent assay.Results In sensitized mast cells,the expression of antigen proteins is mainly in the form of inclusion body,which is successfully purified.After pET-32a(+)-IgE Fc acts on unsensitized RBL-2H3 cells,the cell membrane of RBL-2H3 cells is smooth,and the cells are closely attached to the cell wall,and the shape is spindle.Conclusion PET-32a(+)-IgE Fc can inhibit the degranulation of mast cells,but does not affect the activity of sensitized mast cells.It provides a theoretical basis for the study of allergic diseases such as IgE Fc and asthma.