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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 LyteFDP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。
Objective To prepare monoclonal antibodies (mAb) for the DR segment of sodium-potassium ATPase (Na ~ + -K ~ + -ATPase) α1 subunit DR (897DVEDSYGQQWTYEQR911) and identify its activity. Methods BALB / c mice were immunized with synthetic KL-KLH, and the hybridoma cell lines stably secreting anti-DR were established by cell fusion technique to prepare Na ~ + -K + ATPase alpha 1 subunit DR mAb. The McAb titer, dot immunofluorescence, Western blot and immunofluorescence cytochemistry were used to detect the specificity of the monoclonal antibody in the hybridoma cell supernatant by ELISA. The sensitivity of Na ~ + -K was detected by Senso Lyte FDP protein phosphatase quantitative kit ~ + -ATPase activity, high glucose cell injury assay DR mAb on high glucose-induced cell injury. Results Three stable positive cell lines secreting antibodies were successfully prepared. Select strong positive DRm217 cell line, preparation of ascites further verification. Immunofluorescence staining, immunofluorescence and Western blot showed that the DRm217 mAb could specifically bind to the membrane surface and recognize the DR polypeptide of Na ~ + -K ~ + -ATPase. DRm217 can increase the activity of Na ~ + -K ~ + -ATPase, and has some protective effect on cell injury caused by high glucose. Conclusion The mAb against Na ~ + -K ~ + -ATPase DR was successfully prepared.