鸭疫里默氏杆菌表面抗原D15截短蛋白单克隆抗体的制备及鉴定

Generation and identification of monoclonal antibodies against recombinant tD15 protein of Riemerella anatipestifer

为制备抗鸭疫里默氏杆菌(RA)tD15蛋白的单克隆抗体并对其特性进行鉴定,应用纯化复性的重组tD15蛋白免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0细胞融合,应用有限稀释法筛选出抗RA tD15重组蛋白的阳性杂交瘤细胞株;采用腹水诱生法制备单克隆抗体腹水,用饱和硫酸铵法纯化腹水抗体;采用间接ELISA筛选阳性杂交瘤细胞以及测定单克隆抗体的特性、效价及其相对亲和力。结果显示,获得2株抗RA tD15蛋白的阳性杂交瘤细胞株,遂被命名为4#、16#,2株细胞株经反复冻存复苏,体外传代仍能稳定分泌抗体;4#单克隆抗体属于IgG1,κ链;16#单克隆抗体属于IgG2b,κ链;4#杂交瘤细胞培养上清的效价为1∶3 200,腹水效价为1∶12 800,16#杂交瘤细胞培养上清的效价为1∶6 400,腹水效价为1∶204 800;2株单克隆抗体均能够识别大小约为34ku的蛋白,说明这2株单克隆抗体是特异性识别tD15蛋白的抗体;这2株单克隆抗体能够与鸭疫里默氏杆菌全菌裂解蛋白发生特异性反应,而不与其他鸭源的病原体发生反应;测得它们的亲和常数分别为1.52×109 L/mol和1.00×1010 L/mol。结果表明,这2株单克隆抗体可能识别两种不同的抗原表位。

To prepare monoclonal antibody(McAb)against Riemerella anatipestifer(RA)tD15protein and characterize its biological features,female BALB/c mice were immunized with RA tD15 protein which was purified and refolded.Then splenocytes from the immunized mice were fused with SP2/0-Ag-14,and the hybridoma cell colonies were harvested by limited dilution.Induced ascites methods were used to prepared McAb against RA tD15 protein,and saturated ammonium sulfate was used to depurate the ascites.Indirect ELISA was used to screen the hybridoma cells by determining the specificity,titers and relative affinities of the produced McAbs.The subtypes of IgG were identified using a monoclonal antibody subclass determination kit.We got two strains hybridoma cell lines steadily secreting RA tD15 protein specific McAbs,named 4#and 16#.The subtype of heavy and light chains of the 4# was IgG1andκ,while 16# was IgG2 b andκ.The indirect ELISA titers of these in culture supernatant were 1∶3 200 and 1∶6 400,and McAbs ascites were1∶12 800 and 1∶204 800 respectively.Western-blot analysis revealed that the reacted band was around 34 ku which was identical to the predicted size of RAtD15.The purified McAbs showed strong reactivity only with the RA,and the newly developed McAbs did not show any cross reactivities.The affinity constant of McAb from 4# was 1.52×109L/mol,while 16# was 1.00×1010 L/mol.Two different antigenic epitopes might be recognized by the McAbs.