抗鸭坦布苏病毒NS5蛋白单克隆抗体的制备及鉴定  

Preparation and identification of monoclonal antibody against NS5 protein of duck Tembusu virus

作　　者：杨国平[1] 刁有祥[1] 赵丹丹[1] 陈浩[1] 提金凤[1] 张璐[1] 张英[1] 李川川[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271017

出　　处：《西北农林科技大学学报（自然科学版）》2016年第10期1-6,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31272583);山东自然科学基金项目(ZR2012CM002);国家水禽产业技术体系项目(CARS-43-10)

摘　　要：【目的】制备抗鸭坦布苏病毒(Duck Tembusu virus,DTMUV)NS5蛋白的单克隆抗体。【方法】将实验室保存的PET28a-NS5载体转化入Rosetta(DE3)感受态细胞中进行诱导表达,利用纯化的鸭坦布苏病毒NS5蛋白作为抗原免疫BALB/c小鼠,应用淋巴瘤细胞杂交技术结合有限稀释法制备单抗,并对单抗的特异性、反应性及其抗原表位和亚类进行鉴定。【结果】筛选获得2株稳定分泌针对DTMUV的NS5蛋白单抗的杂交瘤细胞,分别命名为A4D1和B4B8。此2株杂交瘤细胞诱导BALB/c小鼠的抗体效价均可达到1∶64 000。间接ELISA、Western blot和间接免疫荧光(IFA)检测结果表明,所获细胞分泌的抗体能与DTMUV及纯化的NS5蛋白发生特异性反应。经鉴定2株单抗的亚类均为IgG1型,通过叠加ELISA方法,初步判定2株单抗识别不同的抗原表位。【结论】成功获得了抗鸭坦布苏病毒NS5蛋白的单克隆抗体,为研究NS5蛋白的功能奠定了基础。【Objective】This study prepared a monoclonal antibody（McAb）against NS5 protein of duck Tembusu virus（DTMUV）.【Method】PET28a-NS5 vector was transformed into Escherichia coli Rosetta（DE3）,and BALB/c mice were immunized by the purified protein NS5 of DTMUV.Then,the specificity,reactivity,epitope and subclasses were identified.【Result】Two monoclonal antibodies A4D1 and B4B8were obtained by lymphocyte hybridoma technique.The ascites antibody titers of A4D1 and B4B8were both 1∶64 000.Indirect ELISA,Western blot and indirect immunofluorescence assay（IFA）showed that the two McAbs reacted specifically with DTMUV and purified NS5 protein.The subtypes of both McAbs were IgG1 and they can recognize different antigen epitopes based on superposition ELISA method.【Conclusion】Monoclonal antibodies against DTMUV NS5 were successfully prepared,which provides basis for further study of DTMUV NS5 functions.

关 键 词：鸭坦布苏病毒 NS5蛋白 单克隆抗体 

分 类 号：S852.657[农业科学—基础兽医学]