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目的 制备α catenin多克隆抗体 ,为深入研究其功能和探讨其与肿瘤等疾病的相关性提供工具。方法 PCR扩增编码人α cateninC 端 45 5个氨基酸的DNA片段 ,DNA重组入原核表达质粒pGEX 4T 3,转化大肠杆菌JM10 9菌株 ,异丙基 β D硫代半乳糖苷 (IPTG)诱导产生GST α catenin融合蛋白。GST Sepharose亲和层析纯化可溶性融合蛋白 ,免疫新西兰白兔 ,制备抗血清。通过ELISA、免疫荧光细胞化学和SP免疫组织化学法鉴定血清特异性和效价。 结果 成功地构建了pGEX 4T 3 α Cat表达载体 ,转化JM10 9后可高效表达融合蛋白GST α catenin(α Cat)。免疫兔产生的α Cat多抗可特异检测人体组织和细胞中α Cat表达及定位情况。 结论 α catenin抗体效价高 ,特异性强 ,适合免疫组织化学、ELISA等检测应用
Objective To prepare polyclonal antibodies to α catenin and to provide tools for further study of its function and its relationship with tumors and other diseases. Methods A 455-amino acid DNA fragment encoding human α catenin C was amplified by PCR. The recombinant plasmid was transformed into E. coli JM10 9 and induced by IPTG to produce GST α catenin fusion protein. Purified soluble fusion protein by GST Sepharose affinity chromatography and immunized New Zealand white rabbits to prepare antiserum. Serum specificity and titer were identified by ELISA, immunofluorescent cytochemistry and SP immunohistochemistry. Results The pGEX 4T 3α Cat expression vector was successfully constructed. The fusion protein GST α catenin (α Cat) was efficiently expressed after JM109 transformation. Α Cat polyclonal antibodies produced by immunized rabbits specifically detect α Cat expression and localization in human tissues and cells. Conclusion The α catenin antibody has high titer and specificity and is suitable for the detection of immunohistochemistry and ELISA