摘要
为了制备犬瘟热病毒(CDV)核衣壳蛋白(NP)的单克隆抗体,从而为建立CDV的诊断方法提供有效试剂。将纯化后的CDV核蛋白作为免疫原注射至BALB/c小鼠腹腔中,取经4次长程免疫和1次加强免疫并达到融合标准的小鼠脾脏刮制成悬液,与小鼠骨髓瘤细胞(SP2/0)进行融合制备杂交瘤细胞,利用间接ELISA筛选,并对单抗进行生物特性鉴定。结果表明:经过筛选后共获得2株可持续分泌抗CDV NP的单克隆细胞株,以3-3G、1-10B命名。2株细胞株抗体经效价测定均可达到1∶2 048 000;经亚型鉴定,2株杂交瘤细胞均为IgG2型;经过protein A柱纯化,抗体纯度达到了90%以上;BCA法测得抗体浓度至少达2.78 mg/mL;通过Western blot鉴定,制备所得CDV NP单克隆抗体与纯化的CDV NP抗原在预期的60 kd处有蛋白免疫印迹条带。该研究制备的单克隆抗体为后续犬瘟热的检测方法的研究奠定了基础。
In order to prepare the monoclonal antibody against Nucleocapsid Protein(NP) of Canine Distemper Virus(CDV),and to provide an effective reagent for establishing the diagnostic method of CDV,the purified CDV NP is injected into the abdominal cavity of BALB/c mice as the immunogen.The spleen of each mouse is scraped into suspension,when the fusion standard is reached after 4 times of long-term immunization and 1 time of enhanced immunization.Then,fused with mouse myeloma cells(SP2/0) to produce hybridoma cells,these cells are screened by indirect ELISA and their biological characteristics are identified.The results show that,after screening,two monoclonal cell lines which can continuously secrete anti CDV NP are obtained,namely 3-3 G and 1-10 B.The antibody titers of the two cell lines reach 1∶2 048 000.Through subtype identification,both hybridoma cells are IgG2 type.After protein A column purification,the purity of antibodies reaches more than 90%.The antibody concentration measured by BCA method is at least 2.78 mg/mL.Western blots show that the produced CDV NP monoclonal antibodies and purified CDV NP antigens have western blot bands at the expected 60 kd.The monoclonal antibodies produced in this study provide an effective reagent for the subsequent study of detection methods of canine distemper.
作者
冉皑
凌洪权
曾政
骆璐
吴胜昔
董春霞
李云
陈娅
RAN Ai;LING Hongquan;ZENG Zheng;LUO Lu;WU Shengxi;DONG Chunxia;LI Yun;CHEN Ya(College of Pharmacy and Biological Engineering,Chongqing University of Technology,Chongqing 400054,China;Chongqing Animal Disease Prevention and Control Center,Chongqing 401120,China)
出处
《重庆理工大学学报（自然科学）》
CAS
北大核心
2022年第10期305-310,共6页
Journal of Chongqing University of Technology：Natural Science
基金
重庆市技术创新与应用示范专项项目(cstc2018jscxmszdX0049)
重庆市巴南区科技计划项目(2020TJZ008)
重庆理工大学研究生创新基金项目(lgycx 20202128)
大学生创新创业训练计划项目(2021CX146,2021CX147,2021CX148)
重庆市生猪产业技术体系项目(20211105)。
关键词
犬瘟热病毒
核衣壳蛋白
单克隆抗体
细胞融合
效价测定
canine distemper virus
nucleocapsid protein
monoclonal antibody
cell fusion
titer determination
