狂犬病毒M蛋白多克隆抗体的制备与鉴定  

Preparation and Identification of Polyclonal Antibody against Rabies Virus M Protein

作　　者：黎强 丁祎 刘高文 贾亭 高媛媛 陈芮 宋玉竹[1] 韩芹芹[1] 夏雪山[1] 张金阳[1] LI Qiang;DING Yi;LIU Gaowen;JIA Ting;GAO Yuanyuan;CHEN Rui;SONG Yuzhu;HAN Qinqin;XIA Xueshan;ZHANG Jinyang(Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China)

机构地区：[1]昆明理工大学生命科学与技术学院,云南昆明650500

出　　处：《昆明理工大学学报（自然科学版）》2022年第4期90-96,117,共8页Journal of Kunming University of Science and Technology(Natural Science)

基　　金：国家自然科学基金地区基金项目(81860625)。

摘　　要：为了获得狂犬病病毒(RABV)M蛋白,并制备其对应的多克隆抗体.运用分子克隆技术构建pET-28a-CVS-M原核表达重组载体,并将其转化到E.coli Rosetta(DE3)感受态细胞以进行诱导表达.将表达纯化的RABV M重组蛋白免疫小鼠制备多克隆抗体,随后多克隆抗体的效价、特异性以及应用范围通过ELISA和IFA这些实验来检测.结果表明,构建的原核表达重组质粒成功表达了预期的重组蛋白,大小约28 kDa,且能与His-tag多抗血清发生特异性反应.制备的多克隆抗体能够特异性地识别原核M蛋白以及其天然抗原表位,并且其效价高达1∶204800.本研究成功制备了RABV M融合蛋白和较好特异性的RABV M蛋白多克隆抗体,为RABV感染致病机制研究及建立快速、高效免疫学检测技术提供技术指导和材料.The purpose of this study was to purify rabies virus(RABV)M protein and prepare its polyclonal antibody.The prokaryotic expression recombinant plasmid pET-28 a-CVS-M was constructed by molecular cloning technology,and transformed into E.coli Rosetta(DE3)competent cells for induced expression.The purified RABV M fusion protein was used to immunize mice to generate polyclonal antibody,followed by detecting the titer,specificity and application range of the antibody through ELISA and IFA.The results showed that the constructed prokaryotic expression recombinant plasmid successfully expressed the expected recombinant protein,with the size around 28 kDa and which could specifically react with His-tag polyclonal antiserum.The prepared polyclonal antibody could specifically recognize prokaryotic M protein and its natural antigen epitope,and its titer was as high as 1∶204800.In this study,RABV M fusion protein and mouse anti-RABV M polyclonal antibody with better specificity were successfully prepared,which provided technical guidance and materials for the study of RABV infection pathogenesis and the establishment of rapid and efficient immunological detection technology.

关 键 词：狂犬病病毒(RABV) M蛋白 原核表达 多克隆抗体 

分 类 号：R392-33[医药卫生—免疫学]