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
袁陈婷 1杨容 2王立玮 2景凌洁 2陈璐阳 2加羊卓玛 2孙小祥 3孙锦秀 1杨欢1
作者信息
- 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
- 折叠
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.
Abstract
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 ∶ 6 400,1 ∶ 400,1 ∶ 12 800 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Key words
Asini Corii Colla/characteristic polypeptides/polypeptide antigens/polyclonal antibodies/Western blot/origin identification引用本文复制引用
基金项目
国家自然科学基金(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(JCZ2022031)
镇江市科技计划(SH2021057)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据