鸡传染性贫血病毒VP2蛋白单克隆抗体的制备及鉴定  

Development and identification of monoclonal antibodies against Chicken Infectious Anemia Virus(CIAV) VP2

作　　者：陈延飞[1] 覃尧[1] 李夏莹[1] 王永强[1] 李晓齐[1] 曹红[1] 郑世军[1] 

机构地区：[1]中国农业大学动物医学院农业生物技术国家重点实验室,北京海淀100193

出　　处：《中国兽医杂志》2015年第4期3-6,9,共5页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金项目(#31272543);现代农业产业技术体系建设专项资金(#NYCYTX-41)

摘　　要：为制备CIAV VP2单克隆抗体,将原核表达的His-VP2融合蛋白作为免疫原免疫BALB/c小鼠。经4次免疫后,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合。将原核表达的GST-VP2融合蛋白作为包被抗原,利用间接ELISA筛选阳性克隆。阳性细胞株经3次亚克隆后,获得3株稳定分泌抗CIAV VP2蛋白的杂交瘤细胞克隆,分别命名为3D7、3B8、2D3。经间接ELISA测定,以上3株杂交瘤细胞腹水效价分别为1:8.0×10~5、1:4.0×10~5,以及1:6.4×10~6,亲和力解离常数分别为7.34×10^(-11)、2.65×10^(-11),以及2.98×10^(-11)。IgG亚类分别为IgG1、IgG2b以及IgG2b。经Western Blot试验证明,3D7株单抗能特异地识别CIAV VP2蛋白,其识别的抗原表位区域为VP2 N-端的20~40 aa。经IFA试验证明,3株单抗均能识别天然的CIAVVP2蛋白。此单抗为研究CIAVVP2的生物学功能和CIAV致病机理奠定基础。In order to develop monoclonal antibodies （MeAbs）against CIAV VP2, we vaccinated BALB/c mice with His-VP2 protein.An indirect ELISA using GST-VP2 protein as coating antigen was established to screen positive clones. We obtained 3 hybridoma cellclones that stably secreted McAbs against VP2 and these clones were named 3D7,3B8 and 2D3, respectively.The titers in ascite fluidwere 1∶8.0＆#215; 105、1∶4.0x10^5 respectively.The dissociation constants （Kds）were 7.34x10-1, 2.65x10-11, 2.98x10-1, respectively.The isotypes were determined to be IgG1, IgG2b and IgG2b.The McAb 3D7 specifically bound to VP2 and its binding domain was locatedbetween 20 to 40 aa of VP2as demonstrated by Western Blot. These McAbs specifically recognized VP2 in CIAV-infected MDCC-MSBlceUs as examined by Indirect Fluorescence Antibody assay.These McAbs would help to analyze the biological activities of CIAVVP2 and elucidate the mechanism of CIAV infection.

关 键 词：鸡传染性贫血病毒 VP2蛋白 原核表达 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]