禽网状内皮组织增殖病病毒gp90蛋白单克隆抗体的制备及其识别区分析  

Development of monoclonal antibodies against the gp90 protein of reticuloendotheliosis virus and mapping of their recognition regions

作　　者：孙明铭 李晓齐[1] 曹红[1] 王永强[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,北京100193

出　　处：《生物工程学报》2015年第1期75-85,共11页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31272543);现代农业产业技术体系建设专项资金(No.NYCYTX-41)资助~~

摘　　要：为了制备禽网状内皮组织增殖病病毒(REV)gp90蛋白的单克隆抗体,应用His-gp90融合蛋白免疫BALB/c小鼠,取免疫鼠的脾细胞与骨髓瘤细胞(SP2/0)进行融合,经过筛选、3次亚克隆后获得3株稳定分泌抗REV-gp90蛋白的单克隆抗体杂交瘤细胞株,分别命名为3G5-B8、3G5-A10和1G12。经间接ELISA(Enzyme-linked immunosorbent assay)方法检测,单克隆抗体的亲和力解离常数(Kd)分别为6.483×10–10、4.844×10–10和9.330×10–10,3株单抗的亚型分别为Ig G1、Ig G1和Ig G2b。经Western blotting和间接免疫荧光实验检测,3株单抗均能识别REV感染DF-1细胞后产生的gp90蛋白。以Western blotting方法利用单抗检测不同截短的gp90蛋白,初步确定3G5-B8和3G5-A10 2株单抗抗原识别区均位于gp90蛋白第200-245位氨基酸,而1G12株单抗识别区包含第230-235位氨基酸。这些单抗为REV的诊断和致病机理研究奠定了基础。In order to develop monoclonal antibodies （McAbs） against the gp90 protein of reticuloendotheliosis virus （REV）, the His-tagged gp90 protein of REV was used to immunize BALB/c mice. Hybridomas were generated by fusing mouse myeloma cells SP2/0 with the splenocytes from the immunized mice. After screening and 3 rounds of cloning process, 3 hybridomas （3G5-B8, 3G5-A10 and 1G12） that stably secreted McAbs against the REV-gp90 were obtained. The isotypes of the McAbs were determined to be IgG1, IgG1 and IgG2b. The McAbs specifically bound to gp90 in REV-infected DF-1 cells, as demonstrated by Western blotting and indirect immunofluorescence assay. The recognition regions on gp90 that were recognized by 3G5-B8/3G5-A10 and 1G12 were located between amino acids 200 to 245 and 230 to 235, respectively, as demonstrated by Western blotting analysis. These McAbs will be useful in the diagnosis and pathogenesis study of REV.

关 键 词：禽网状内皮组织增殖病病毒 gp90蛋白 单克隆抗体 亲和力 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]