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

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.