摘要
目的应用制备纯化的鸭坦布苏病毒(DTMUV)非结构蛋白1(NS1),制备其单克隆抗体(mAb)并鉴定抗体的特异性。方法将纯化的NS1蛋白免疫BALB/c小鼠,聚乙二醇(PEG)处理小鼠脾细胞与Sp2/0细胞融合,经ELISA筛选和亚克隆得2株NS1单克隆抗体细胞株,命名为NS1B、NS1E。利用ELISA测定NS1 mAb效价,Western blot法和间接免疫荧光法(IFA)鉴定NS1 mAb特异性,试剂盒测定NS1mAb亚类。最后,用NS1 mAb对感染DTMUV后6、12、24、36 h的BHK-21细胞进行IFA检测,观察NS1蛋白的表达。结果NS1 mAb的亚型为IgG1,效价达到1∶1.28×10^(4);表明制备的NS1 mAb能识别天然NS1蛋白;DTMUV感染BHK-21细胞12 h后NS1蛋白开始表达。结论获得2株具有反应性、特异性且效价高的NS1 mAb,可用于DTMUV感染检测。
Objective To prepare the monoclonal antibody(mAb)against the nonstructural protein 1(NS1)of duck Tembusu virus(DTMUV)and identify its specificity.Methods The recombinant NS1 protein was purified and used to immunize BALB/c mice to prepare mAb.Splenocytes isolated from the immunized mice were hybridized with Sp2/0 myeloma cells using the polyethylene glycol method.Indirect ELISA was used for screening mAb-secreting hybridomas and detecting mAb titers.Indirect immunofluorescence assay and Western blotting were used to detect the reactivity and specificity of the mAb.Subclass of NS1 mAb was determined using a commercial kit.Finally,the mAb was used to analyze the dynamic distribution of NS1 protein in DTMUV-infected cells at different time points.Results Two hybridoma cell lines named NS1B and NS1E were successfully screened and purified.The subclass of both mAb strains was IgG1 and the titer reaches 1∶1.28×10^(4) for all NS1 mAb.Moreover,both mAbs showed a positive reaction with DTMUV-infected BHK-21 cells.We found BHK-21 cells began to express NS1 protein as early as 12 hours after DTMUV infection.Conclusion Two mAbs against NS1 protein of DTMUV have been successfully prepared,which is a useful tool for DMTUV diagnosis and treatment.
作者
顾香雪
周祺
曹昳
夏宇航
张冲
王桂军
GU Xiangxue;ZHOU Qi;CAO Yi;XIA Yuhang;ZHANG Chong;WANG Guijun(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control,Hefei 230036,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2021年第5期455-459,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点研发计划(2018YFD0500100,2016YFD0500805)。
