摘要
目的 制备和纯化兔抗小鼠βcatenin抗体,分析其在乳腺癌细胞和乳腺良、恶性组织中的表达。方法 应用重组小鼠βcatenin蛋白,常规免疫雄性新西兰兔制备抗βcatenin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βcatenin抗体的纯度和特异性以及乳腺癌细胞株MCF 7和T 4 7D中βcatenin蛋白的表达。用免疫组织化学SP法检测βcatenin在正常乳腺组织和乳腺癌组织中的不同表达。结果 用重组小鼠βcatenin蛋白免疫新西兰兔6周后,应用间接ELISA法检测静脉血中βcatenin抗体效价为1∶2 0 4 80 0 (A4 50nm =1.0 1)。将βcatenin经SDS PAGE分离,可见1条相对分子质量(Mr)为92 0 0 0的蛋白条带,与文献报道相符。用抗βcatenin血清做Westernblot分析显示,在Mr为92 0 0 0处有1条明显的区带,证明是抗βcatenin特异性抗体。从人乳腺癌细胞株MCF 7和T 4 7D提取全细胞蛋白,经用所制备的βcatenin抗体反应,均观察到βcatenin蛋白的表达。人乳腺组织切片经抗βcatenin抗体免疫组化染色,在正常乳腺组织细胞膜中可见棕黄色颗粒,而在乳腺癌组织的细胞浆或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βcatenin抗体。用该抗体检测证实,正常乳腺组织βcatenin表达在细胞膜中。
Objective To prepare and purify rabbit antibody and analysis its application in breast cancer diagnosis. Methods The anti-β-catenin antibody was prepared by immunizing rabbits with recombinant mouse β-catenin protein. The purity and specificity of the antibody were analyzed by Western blotting. The expressions of β-catenin in human breast cancer cell lines and in breast tissue were analysis by immunoblotting and immunohistochemical staining. Results The titer of anti-β-catenin antibody in the serum was 1∶204 800 (A 450 nm = 1.01) after immunization with β-catenin for 6 weeks. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed that there was an obvious protein band with M r 92 000, which confirmed the specificity of obtained anti-β-catenin antibody. The expressions of β-catenin were detected by Western blotting in human breast cancer cell lines MCF-7 and T-47D. The β-catenin was expressed in cytomembrane of normal breast tissue and in cytoplasm and nucleolus of breast cancer tissue. Conclusion The rabbit anti-β-catenin antibody with high titer and specificity is prepared. The β-catenin is expressed in cytomembrane of normal breast tissue and in cytoplasm and nucleolus of breast cancer tissue. The results may be favorable for clinical diagnosis of breast cancer.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第2期117-120,共4页
Immunological Journal
基金
美国MD技术公司合作课题基金资助项目 (2 0 0 2年 )
