鸡传染性贫血病毒VP2蛋白单克隆抗体的研制及其识别抗原表位的鉴定  

Development and Epitope Identification of a Monoclonal Antibody against VP2 Protein of Chicken Infectious Anemia Virus

作　　者：汪铭锐 吴俊花 王飞[1,2] 邵红霞 钱琨[1,2,3] 叶建强[1,2,3,4] 秦爱建[1,2,3,4] WANG Mingrui;WU Junhua;WANG Fei;SHAO Hongxia;QIAN Kun;YE Jianqiang;QIN Aijian(Ministry of Education Key Laboratory for Avian Preventive Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;Jiangsu Province Key Laboratory of Preventive Veterinary Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China)

机构地区：[1]扬州大学兽医学院教育部禽类预防医学重点实验室,扬州225009 [2]扬州大学兽医学院江苏省动物预防医学重点实验室,扬州225009 [3]扬州大学兽医学院江苏高校动物重要疫病与人兽共患病防控协同创新中心,扬州225009 [4]扬州大学教育部农业与农产品安全国际合作联合实验室,扬州225009

出　　处：《畜牧兽医学报》2022年第2期597-606,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划(2016YFD0500803)。

摘　　要：拟制备针对鸡传染性贫血病毒(CIAV)VP2蛋白的单克隆抗体(mAb),为CIAV的诊断和病毒生物学特性研究提供有用制剂。以PCR技术扩增CIAV VP2基因并克隆到原核表达载体pET-32a中,经IPTG诱导表达。以原核表达的融合蛋白免疫BALB/c小鼠,利用杂交瘤技术研制并筛选分泌抗CIAV VP2蛋白mAb的阳性杂交瘤细胞;采用截短表达CIAV VP2基因方法鉴定mAb识别的抗原表位。结果显示:成功获得了1株能稳定分泌抗CIAV VP2蛋白mAb的杂交瘤细胞株,并命名为CIAV-VP2-4A12;亚类鉴定结果表明,该mAb重链为IgG_(1)型,轻链为kappa链;Western blot结果显示,该mAb可以识别大肠杆菌,Sf9细胞和转染pCAGGS-VP2-Flag的DF1细胞中表达的VP2蛋白。使用原核表达截短蛋白进行表位鉴定,发现该mAb识别抗原表位序列是^(155)KTVRW^(159),序列比对发现该表位在CIAV中高度保守。本研究成功制备了针对CIAV VP2蛋白的特异性mAb,并精确鉴定了其识别表位,为VP2蛋白功能的研究以及CIAV的诊断方法研发提供了有效工具。The aim of this study was to prepare monoclonal antibody(mAb)against VP2 protein of chicken infectious anemia virus(CIAV)for diagnosis of CIAV and further research in characteristics of CIAV.The VP2 gene of CIAV was amplified and cloned into the pET-32a plasmid.The VP2 fusion protein was expressed in Escherichia coli induced by IPTG.Hybridoma cells were fused between SP2/0 cells and spleen cells of BALB/c mice immunized with the expressed protein and screened by immunofluenscence assay.The epitope recognized by mAb was further identified by the truncated VP2 proteins in Western blot.One hybridoma cell line named CIAV-VP2-4A12 that can stably secrete mAb against CIAV VP2 protein was successfully obtained.The isotype of the mAb was IgG1 and the light chain was kappa chain.Western blot assay showed that mAb could react with the VP2 protein expressed in Escherichia coli,Sf9 cells or DF1 cells transfected with pCAGSS-VP2-Flag.The epitope recognized by the mAb CIAV-VP2-4A12 was identified as ^(155)KTVRW^(159) of VP2,which sequence is highly conserved among different CIAV strains.In this study,a monoclonal antibody against CIAV VP2 was successfully developed and the antigenic epitope recognized by the mAb was identified,which will be useful tool for research on the function of the VP2 protein and development of diagnostic methods for CIAV.

关 键 词：鸡传染性贫血病毒 VP2蛋白 单克隆抗体 抗原表位 

分 类 号：S852.659.2[农业科学—基础兽医学] S852.4[农业科学—兽医学]