抗鸡贫血病毒结构蛋白单克隆抗体制备及其对应抗原表位分析  

Development of Monoclonal Antibodies Against VP1 Protein of Chicken anemia virus and Its Identification of Epitopes

作　　者：王晓艳[1] 高宏雷[1] 高玉龙[1] 程宇[2] 王笑梅[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽传染病实验室,哈尔滨150001 [2]新疆农业大学,乌鲁木齐830052

出　　处：《农业生物技术学报》2007年第4期557-561,共5页Journal of Agricultural Biotechnology

基　　金：国际合作项目(No.2003DF010012)资助

摘　　要：以纯化的重组鸡贫血病毒结构蛋白为抗原,免疫6～8周龄的雌性Balb/c鼠,经过3次免疫后,取其脾细胞与SP2/0骨髓瘤细胞进行融合,共获得6株能稳定分泌特异性抗体的阳性细胞株。间接免疫荧光实验证实,6株单抗与鸡贫血病毒(Chicken anemia virus,CAV)感染的MDCC-MSB1细胞发生特异性反应。Western blot结果表明,单抗与杆状病毒表达的VP1重组蛋白发生特异性反应。单克隆抗体亚型鉴定结果表明,6株单抗均为IgG1亚型,轻链均为κ链。用单克隆抗体对分段表达的VP1蛋白进行免疫印迹分析,鉴定出3个抗原表位区,分别位于VP1的218～274氨基酸(aa)以及275～301aa和324～369aa之间。Female Balb/c mice of 6-8 weeks were immunized by the purified recombinant Chicken amemia virus （CAV） fusion protein. After 3 immunizations, the spleen cells frOln immunized mice were fused with mouse myeloma cells SP2/0. Six positive hybridoma cell lines stably secreting monoclonal antibodies （MAbs） were selected by 3 cycles of limited dilutions. ,All MAbs reacted with MDCC-MSB1 cell infected with CAV by indirect immunofluorescence assay （IFA） and they recognized the VP1 protein expressed in Baculovirus by Western blot. It demonstrated that all MAbs possesed good specificity. The 6 MAbs were IgG1 subtype and light chains of all MAbs were kappa. Taking advantage of VP1 fragments expressed, the epitopes corresponding to the MAbs were analyzed. The results showed that the monoclonal antibodies 1C5,2F3,4D2 and 4E8 recognized the same epitope, which was located in 218-274 aa. Moreover, the two other epitopes were identified and they were located in 274-301 aa and 324-369 aa, respectively.

关 键 词：鸡贫血病毒(CAV) 结构蛋白(VP1) 单克隆抗体 抗原表位 

分 类 号：S188[农业科学—农业基础科学]