【摘要】 目的制备钠钾ATP酶(Na~+-K~+-ATPase)α1亚基DR区段(897DVEDSYGQQWTYEQR911)的单克隆抗体(mAb)并鉴定其活性。方法以合成的DR-钥孔戚血蓝素(KLH)为免疫原,免疫BALB/c小鼠,通过细胞融合技术建立能稳定分泌抗DR的杂交瘤细胞株,制备Na~+-K~+-ATPaseα1亚基DR mAb。ELISA测定杂交瘤细胞上清中mAb效价,斑点免疫杂交、Western blot法及免疫荧光细胞化学染色法检测单克隆的特异性,采用Senso LyteFDP蛋白磷酸酶定量试剂盒检测Na~+-K~+-ATPase活性,高糖细胞损伤实验检测DR mAb对高糖引起细胞损伤的保护作用。结果成功制备3株稳定分泌抗体的阳性细胞株。选择强阳性DRm217细胞株,制备腹水进一步验证。免疫荧光细胞化学染色、免疫斑点杂交及Western blot法结果显示,DRm217 mAb能够特异性结合在细胞膜表面,识别Na~+-K~+-ATPase的DR多肽。DRm217能够提高Na~+-K~+-ATPase活性,对高糖引起的细胞损伤具有一定的保护作用。结论成功制备了针对Na~+-K~+-ATPase DR多肽的mAb。更多还原

【Abstract】 Objective To prepare monoclonal antibodies against DR region( 897DVEDSYGQQWTYEQR911) of Na~+-K~+-ATPase α1 subunit and identify their properties. Methods BALB / c mice were immunized with DR-keyholelimpet hemocyanin( KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2 /0 mouse myeloma cells.Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting,Western blotting and immunofluorescence assay. Na~+-K~+-ATPase activity was detected by Senso Lyte FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line,named DRm217,was selected to prepare ascites. Dot blotting,Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na~+-K~+-ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na~+-K~+-ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na~+-K~+-ATPase α1 subunit is prepared.更多还原