Avi-tag-MrgprX2新型CMC固定相制备与中药注射剂中类过敏组分分析

中药注射剂引起类过敏反应的物质基础是中药注射剂质量控制的重要内容。细胞膜色谱技术已被广泛应用于中药物质基础研究，基于野生型肥大细胞或MrgprX2受体过表达细胞的传统细胞膜色谱模型能够有效分析中药注射剂中的类过敏组分，但均存在稳定性差、特异性低的缺点，降低了对类过敏组分辨识的准确性及高效性。本课题拟构建Avi-tag-MrgprX2融合蛋白过表达的HEK293细胞，合成生物素键合材料，通过Avi-tag的生物素化定向固定MrgprX2受体，制备Avi-tag-MrgprX2新型固定相，有效提高固定相稳定性及对类过敏组分识别的特异性。构建Avi-tag-MrgprX2细胞膜色谱模型，并与高效液相色谱质谱联用构成二维分析系统，对中药注射剂中的类过敏组分进行精准分析。本项目制备的固定相能够实现对类过敏组分的高精度、高准确度分析，对中药制剂安全性评价具有重要的理论意义及应用价值。

The substantial basis of anaphylactoid reactions caused by traditional Chinese medicine injections is important for quality control of traditional Chinese medicine injections. Cell membrane chromatography technology has been widely used in the study of substantial basis. Combined with liquid chromatography-mass spectrometry, CMC can effectively screen, analyze, and further identify the active components related to anaphylactoid reactions in traditional Chinese medicine injections. Previous commonly used cell membrane chromatography models based on wild-type mast cells or MrgprX2 receptor overexpressed cells can effectively analyze allergic components in traditional Chinese medicine injections, but they all have shortcomings of poor stability and specificity, thus reducing the accuracy and efficiency of receptor-targeted identification for allergic components. This project intends to construct HEK293 cells overexpressing Avi-tag-MrgprX2 fusion protein, synthesize biotin-bonded carrier material, and achieve oriented/site-specific immobilization of Avi-tag-MrgprX2 cell membrane biomaterials through Avi-tag biotinylation. Hopefully, the subsequently prepared Avi-tag-MrgprX2 immobilized new stationary phase can effectively enhance the stability of the stationary phase, improve the specificity of the chromatography model to allergic components. The Avi-tag-MrgprX2 cell membrane chromatography model is constructed and combined with high performance liquid chromatography mass spectrometry to form a two-dimensional analysis system for the purpose of achieving accurate analysis of allergic components in traditional Chinese medicine injections. The new stationary phase prepared in this project can realize high-precision and high-accuracy analysis for allergic components, also has important theoretical significance and application value for the safety evaluation of traditional Chinese medicine preparations.