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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。
Objective To obtain a group of anti-human CD130 intracellular phosphorylated tyrosine monoclonal antibody mAb , for the study of IL-6-mediated signal transduction. Methods According to the amino acid sequence of the intracellular N752-G766 region of CD130, Y159 phosphorylated 15 peptide was chemically synthesized as antigen. A series of anti-CD130 phosphorylated tyrosine mAbs were prepared by cell fusion method. ELISA, Western blot, immunoprecipitation and Immunohistochemistry and other techniques, the nature of this group of mAb and function were discussed. Results (1) Four hybridoma cell lines (MIY1, MIY2, MIY3 and MIY4) that stably secreted anti-CD130 intracellular Y759 phosphorylated mAbs were obtained by cell fusion. The mAbs produced specific reaction only to the synthetic peptide N752-G766 . (2) Western blot analysis showed that the relative molecular mass of the target molecule recognized by this group of mAbs was 13 × 104-14 × 104. Mr antibodies immunoprecipitated with this group of mAbs were 13 × 104-14 × 104 proteins recognized by anti-CD130 mAb while 13 × 104-14 × 104 molecules immunoprecipitated with anti-CD130 mAb were also recognized by this group of mAbs; ( 3) Further studies confirm that this group of mAbs recognizes tyrosine phosphorylated protein molecules induced by IL-6. Conclusion The anti-human CD130 intracellular phosphorylated tyrosine mAb was successfully developed.