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目的 :制备并纯化兔抗小鼠脾信号转导和转录激活因子 5(STAT5 )的抗体 ,分析其在乳腺癌细胞以及良恶性乳腺组织中的表达。方法 :应用重组小鼠STAT5蛋白 ,常规免疫雄性新西兰兔制备抗STAT5抗体 ,并用盐析法和离子交换层析技术进行纯化。用免疫印迹法 (Westernblot)检测抗STAT5抗体的纯度和特异性 ,以及乳腺癌细胞株MCF 7和MDA MB 2 31中STAT5蛋白的表达 ;用免疫组化SP法检测STAT5在正常乳腺组织和乳腺癌组织中的表达。结果 :以重组小鼠STAT5蛋白免疫新西兰兔 6wk后 ,用间接ELISA法检测静脉血中STAT5抗体的效价为 1∶2 0 4 80 0 (A450 =1.0 7)。将STAT5经SDS PAGE分离 ,可见 1条相对分子质量 (Mr)为 90 0 0 0～ 95 0 0 0的蛋白带 ,与文献 [3]的报道相符。用抗STAT5血清做Westernblot分析显示 ,在Mr为 90 0 0 0～ 95 0 0 0处也有 1条明显的带 ,证明是抗STAT5的特异性抗体。将人乳腺癌细胞株MCF 7和MDA MB 2 31的细胞浆和细胞核蛋白 ,与所制备的抗STAT5抗体均可起反应。人乳腺组织切片用抗STAT5抗体做免疫组化染色显示 ,在正常乳腺组织细胞浆中可见棕黄色颗粒 ,而在乳腺癌组织的细胞浆和细胞核中可见棕黄色颗粒。结论 :制备了高效价、特异性的兔抗STAT5抗体。用该抗体检测证实 ,STAT5只在正常乳腺组织的细胞
OBJECTIVE: To prepare and purify rabbit anti-mouse spleen signal transducer and activator of transcription 5 (STAT5) antibody and analyze its expression in breast cancer cells and in benign and malignant breast tissues. Methods: Recombinant mouse STAT5 protein was used to immunize New Zealand white rabbits to prepare anti-STAT5 antibody and purified by salting out and ion exchange chromatography. The purity and specificity of anti-STAT5 antibody and the expression of STAT5 protein in breast cancer cell lines MCF 7 and MDA MB 2 31 were detected by Western blotting. The expression of STAT5 in normal breast tissue and breast cancer Expression in tissue. RESULTS: After immunized New Zealand rabbits with recombinant mouse STAT5 protein for 6 weeks, the titer of STAT5 antibody in venous blood by indirect ELISA was 1: 24080 (A450 = 1.07). STAT5 was separated by SDS PAGE. One protein band with relative molecular mass (Mr) of 90 0 0 0 ~ 95 0 0 0 was observed, which accorded with the reported in [3]. Western blot analysis using anti-STAT5 serum showed that there was also a clear band at Mr 90 0 0 0 0 0 0 0 0 0 0 0 0 0 which proved to be an anti-STAT5 specific antibody. The cytoplasm and nucleoprotein of human breast cancer cell lines MCF7 and MDA MB231 were reacted with the prepared anti-STAT5 antibody. Immunohistochemical staining of human mammary gland sections with anti-STAT5 antibody revealed brownish-yellow granules in the cytoplasm of normal breast tissue and brownish-yellow granules in the cytoplasm and nucleus of breast cancer tissues. Conclusion: High titer and specificity rabbit anti-STAT5 antibody was prepared. With this antibody test confirmed that, STAT5 only in normal breast tissue cells