基于特征多肽抗原的阿胶基原鉴定  

Origin identification of Asini Corii Colla based on characteristic polypeptide antigens

作　　者：袁陈婷 杨容 王立玮 景凌洁 陈璐阳 加羊卓玛 孙小祥 孙锦秀[1] 杨欢 YUAN Chen-ting;YANG Rong;WANG Li-wei;JING Ling-jie;CHEN Lu-yang;Jia-yang-zhuo-ma;SUN Xiao-xiang;SUN Jin-xiu;YANG Huan(Department of Pharmacy,The People’s Hospital of Rugao,Rugao 226500,China;School of Pharmacy,Jiangsu University,Zhenjiang 212013,China;Department of Pharmacy,The Affiliated Zhenjiang Hospital of Nanjing University of Chinese Medicine,Zhenjiang 212003,China)

机构地区：[1]如皋市人民医院药剂科,江苏如皋226500 [2]江苏大学药学院,江苏镇江212013 [3]南京中医药大学附属镇江医院药剂科,江苏镇江212003

出　　处：《中成药》2024年第4期1214-1219,共6页Chinese Traditional Patent Medicine

基　　金：国家自然科学基金项目(81773855);北京康盟慈善基金会卫生健康发展促进项目(KM-ZLGJ-009);南通市基础科学研究和民生科技计划项目(JCZ2022031);镇江市科技计划项目(SH2021057)。

摘　　要：目的 以特征多肽为半抗原并制备多克隆抗体,鉴定阿胶原材料基原。方法 经文献调研及数据库比对筛选特征多肽,将多肽抗原与血蓝蛋白偶联并免疫Balb/c小鼠制备多克隆抗体,通过ELISA、dot blot和Western blot分析与验证多克隆抗体的产生、效价及特异性。结果 筛选所得5个特征多肽序列均具有T细胞表位和B细胞表位,亲水性-1~0,二级结构大多为无规则卷曲。多肽抗原ECS1、ECS3~ECS5在二次加强免疫后,效价分别为1∶6 400、1∶400、1∶12 800和1∶800。制备所得的多克隆抗体之间不产生交叉反应,而且多克隆抗体Ab-ECS3与驴皮及其伪品蛋白的结合显现出较大差异。结论 以特征多肽为半抗原制备所得的多克隆抗体可通过Western blot对驴皮进行真伪鉴别,为胶类中药的基原鉴定提供了参考。AIM To identify the origin of the raw materials of Asini Corii Colla by preparing polyclonal antibodies with characteristic polypeptides as antigens.METHODS The characteristic polypeptides were screened by literature research and database comparison.Balb/c mice were immunized with KLH-conjugated polypeptides to prepare polyclonal antibodies.Then,the production,titers and specificities of the polyclonal antibodies were analyzed and verified by ELISA,dot blot and Western blot.RESULTS All the five characteristic polypeptides had T cell and B cell epitopes,with hydrophilicity of(-1-0),and most of the secondary structures were random curling.After the second booster immunization,the titers of Ab-ECS1,Ab-ECS3,Ab-ECS4 and Ab-ECS5 were 1∶6400,1∶400,1∶12800 and 1∶800,respectively.In addition,there was no cross-reaction between the prepared polyclonal antibodies,and the binding of the polyclonal antibody Ab-ECS3 to the donkey skin and its counterfeit proteins showed a large difference.CONCLUSION The polyclonal antibodies prepared with the characteristic polypeptides as hapten can be used to identify the authenticity of donkey skin by Western blot,which provides a reference for the identification of the origin of gelatinous Chinese medicine.

关 键 词：阿胶 特征多肽 多肽抗原 多克隆抗体 Western blot 基原鉴定 

分 类 号：R282.5[医药卫生—中药学]