摘要
制备并鉴定针对多房棘球绦虫(Echinococcus multilocularis)Tetraspanin 5(TSP5)蛋白的单克隆抗体,评估其在包虫病诊断与免疫治疗中的应用潜力。通过基因合成构建pET-28a-TSP5原核表达载体,诱导表达并纯化重组TSP5蛋白;以该蛋白免疫Balb/c小鼠,采用杂交瘤技术筛选阳性杂交瘤细胞株,经有限稀释法进行亚克隆筛选;通过间接ELISA检测抗体效价,采用免疫荧光法分析抗体与天然组织中TSP5蛋白的结合特性,并对抗体可变区序列进行测序与分析。结果显示,本试验成功构建pET-28a-TSP5表达载体,表达并纯化出大小约为8.6 kDa的重组TSP5蛋白(对应其膜外结构域)。经细胞融合与亚克隆筛选,获得一株稳定分泌抗TSP5单克隆抗体的杂交瘤细胞株3B7G12。间接ELISA检测显示该抗体效价达1∶243000,免疫荧光检测结果表明该抗体可特异性识别多房棘球蚴组织中原头蚴与囊壁表面的TSP5蛋白。抗体可变区测序结果显示该抗体重链与轻链CDR区序列明确,具备高亲和力结合潜力。本研究成功制备了高效价、高特异性的抗TSP5单克隆抗体,该抗体能够精准结合多房棘球绦虫组织中的TSP5蛋白,为泡型包虫病的分子诊断提供了新型工具,并为后续CAR-T疗法及抗体药物开发提供了重要依据。
The objective was to prepare and identify a monoclonal antibody(mAb)against Tetraspanin 5(TSP5)of Echinococcus multilocularis,and evaluate its potential application in the diagnosis and immunotherapy of echinococcosis.The prokaryotic expression vector pET-28a-TSP5 was constructed by gene synthesis.Recombinant TSP5 protein was expressed and purified.Balb/c mice were immunized with the protein,and hybridoma cells were screened by hybridoma technology.Positive clones were subcloned by limiting dilution.Antibody titer was detected by indirect ELISA,and binding specificity to native TSP5 in hydatid tissues was analyzed by immunofluorescence.The variable region sequences of the antibody were sequenced and analyzed.The results showed that the pET-28a-TSP5 vector was successfully constructed,and a recombinant TSP5 protein of approximately 8.6 kDa(corresponding to the extracellular domain)was expressed and purified.A hybridoma cell line named 3B7G12,stably secreting anti-TSP5 mAb,was obtained.The antibody titer reached 1∶243000 by ELISA.Immunofluorescence results showed that the mAb specifically recognized TSP5 protein on the surface of protoscoleces and cyst walls in E.multilocularis tissues.Sequencing of the variable regions revealed well-defined CDR sequences with high potential affinity.A high-titer and highly specific anti-TSP5 monoclonal antibody was successfully prepared,which accurately localized TSP5 protein in E.multilocularis tissues.This antibody provides a new tool for molecular diagnosis of alveolar echinococcosis and lays an experimental foundation for the development of CAR-T therapy and antibody-based drugs.
作者
罗琦
程永康
陈思思
常岭迪
李子华
LUO Qi;CHENG Yongkang;CHEN Sisi;CHANG Lingdi;LI Zihua(General Hospital of Ningxia Medical University,Yinchuan Ningxia 750003,China;School of Basic Medical Sciences,Ningxia Medical University,Yinchuan Ningxia 750004,China;Mobile Second Detachment of Qinghai Provincial Corps of the Chinese People’s Armed Police Force,Tongren Qinghai 811300,China)
出处
《中兽医医药杂志》
2025年第5期35-40,共6页
Journal of Traditional Chinese Veterinary Medicine
基金
宁夏回族自治区重点研发计划项目(2022BSB03092)。
作者简介
罗琦(1988-),男,药师,研究方向为药理学和药物治疗学。Email:624340257@qq.com;通信作者:李子华(1987-),男,副教授,博士,研究方向为包虫病和抗体治疗及分子生物学。Email:lizihua012178@126.com。
