猪伪狂犬病病毒HeN1株gB蛋白单克隆抗体的制备及B细胞线性表位鉴定  

Preparation of the monoclonal antibody against the gB protein and identification of the B-cell epitope in gB of pseudorabies virus HeN1 strain

作　　者：赵微[1] 田志军[2] 王倩[2] 倪宏波[1] 彭金美[2] ZHAO Wei;TIAN Zhi-jun;WANG Qian;NI Hong-bo;PENG Jin-mei(Heilongjiang Bayi Agricultural University,Daqing 163319,China;Harbin Veterinay Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]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,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2018年第9期854-857,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2016YFD0500104)

摘　　要：为制备抗猪伪狂犬病病毒(PRV)gB蛋白的单克隆抗体(MAb),本研究将PRV HeN1株全病毒免疫BALB/c小鼠,取免疫后小鼠的脾细胞与骨髓瘤细胞SP2/0融合,用间接免疫荧光方法筛选,获得了一株稳定分泌抗PRV g B蛋白的杂交瘤细胞株1E7。Western blot结果显示,MAb 1E7与PRV全病毒和真核表达的g B蛋白均能够反应。阻断ELISA试验显示MAb 1E7与PRV的结合可以被猪阳性血清阻断。抗体亚类鉴定显示MAb 1E7的重链为Ig G2b亚类,轻链为kappa链。利用大肠杆菌原核表达系统,表达一系列截短的g B蛋白,最终确定1E7识别的抗原表位是^(81)SAEESLE^(87)。序列分析和western blot结果表明该表位在各PRV病毒株间相对保守。该MAb的制备为建立具有广泛适用性检测PRV野毒感染和疫苗免疫效果评价的阻断ELISA方法奠定了基础。To prepare the monoclonal antibody （MAb） against glycoprotein B （gB） of pseudorabies virus （PRV）, BALB/c mice were immunized with PRV HeN1 strain. The spleen cells of the immunized mice were isolated and fused with SP2/0 myeloma cells. One hybridoma cell line （1E7） secreting MAb against gB protein was identified using the irnmunofluorescence assay （IFA）. Western blot analysis showed that MAb 1E7 specifically recognized the gB protein expressed on both PRV and the membrane of eukaryotic cells. ELISA showed that PRV-positive serum was able to inhibit the binding of MAb 1E7 to PRV. The heave and light chains of MAb 1E7 were IgG2b subtype and K chain, respectively. A series of truncated gB proteins were expressed by prokaryotic expression system and the epitope sequence recognized by the MAb 1E7 was identified as 8tSAEESLE87. This epitope was conservative among different PRV strains by both sequence analysis and western blot assay. The MAb 1E7 prepared in this study provides experimental material for the development of PRV antibody detection method for both monitoring virus infection and evaluating the immune effect of vaccines.

关 键 词：猪伪狂犬病病毒 gB蛋白 单克隆抗体 表位 

分 类 号：S852.65[农业科学—基础兽医学]