兔抗STAT5抗体的制备及其在乳腺癌诊断中的应用

Preparation of rabbit anti-STAT5 antibody and its application in breast cancer diagnosis

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 。

AIM: To prepare and purify rabbit antibody against STAT5 and evaluate its application in breast cancer diagnosis. METHODS: The rabbits were immunized with recombinant mouse STAT5 protein for preparing antibody against STAT5. The purity and specificity of the antibody were analyzed by Western blot. The proteins expressed in human breast cancer cell lines and normal breast tissue and breast cancer tissues were analyzed by Western blot and immunohistochemical staining with the anti-STAT5 antibody. RESULTS: The titer of anti-STAT5 antibody in the serum was 1∶204 800 (A 450=1.07) at 6 weeks after being immunized with STAT5. The SDS-PAGE and Western blot analysis showed that there was a obvious protein band with M r 90 000-95 000, confirming the specificity of obtained anti-STAT5 antibody. The expression of STAT5 could be detected with Western blot in both human breast cancer cell lines MCF-7 and MDA-MB-231.Immunohistochemical staining indicated that STAT5 was expressed in cytoplasm of normal breast tissues while in cytoplasm and nucleolus of breast cancer tissues. CONCLUSION: The rabbit anti-STAT5 antibody with high titer and specificity has been prepared. The STAT5 is expressed in cytoplasm of normal breast tissue, and in nucleolus of breast cancer tissue. The preparation of anti-STAT5 antibody provides an important reagent for clinical diagnosis of breast cancer.