抗人CD130胞内磷酸化酪氨酸单克隆抗体的制备与鉴定

The preparation and characterization of monoclonal antibodies against phosphorylated-tyrosine in intracellular region of human CD130

目的获得一组抗人CD130胞内磷酸化酪氨酸单克隆抗体mAb,用于研究IL-6介导的信号转导。方法按照CD130分子胞内N752-G766区的氨基酸序列,化学合成Y759磷酸化的15肽作为抗原,应用细胞融合法制备一组抗CD130磷酸化酪氨酸的mAb,并用ELISA、Westernblot、免疫沉淀和免疫组化染色等技术,对这组mAb的性质和功能进行探讨。结果(1)通过细胞融合获得了4株稳定分泌抗CD130胞内Y759磷酸化mAb的杂交瘤细胞株(MIY1、MIY2、MIY3和MIY4)这组mAb仅对合成肽N752-G766产生特异性反应。(2)Westernblot分析表明这组mAb识别的靶分子的相对分子质量Mr为13×104～14×104的。用此组mAb免疫沉淀的Mr为13×104～14×104蛋白,可被抗CD130mAb所识别;同时用抗CD130mAb免疫沉淀的13×104～14×104分子也可被这组mAb所识别;(3)进一步研究证实这组mAb可识别由IL-6诱导的细胞内酪氨酸磷酸化的蛋白分子。结论成功地研制了抗人CD130胞内磷酸化酪氨酸的mAb。

Aim To obtain the tyrosine-phosphorylated monoclonal antibodies(mAbs)against human CD130 molecules. Methods The peptide(N752-G766)of CD130 was used as the antigen. Hybridoma cell lines were established by cell fusion. The mAbs were characterized by ELISA,Western blot, immunoprecipitation and immunohistochemical staining. Results (1)4 hybridoma cell lines(MIY1,MIY2,MIY3 and MIY4) could secrete stably mAbs which can react specificly to N752-G766 peptide.(2) The results of Western blot showed that mAbs could recognize molecules of relative molecular mass(Mr) 13×104～14×104 in the target cells. The mAbs could immunoprecipitate molecules recognized by anti-CD130 mAb.(3)The molecules of Mr being 13×104～14×104 recognized by the mAbs ro anti CD130 mAb were the intracellular tyrosine-phosphorylated proteins induced by IL-6. Conclusion Antibodies against phosphorylated tyrosine in human CD130 molecules has been prepared and characterized successfully.