小鼠成纤维细胞激活蛋白多克隆抗体的制备及应用

Preparation and application of anti-mouse fibroblast activation protein polyclonal antibody

目的:表达和纯化小鼠成纤维细胞激活蛋白α(FAPα)二肽基肽酶催化核心序列(FAPαc)融合蛋白(His-FAPαc),制备兔抗小鼠FAPα多克隆抗体,并用此抗体检测FAPα在肿瘤细胞和成纤维细胞以及肿瘤间质中的表达情况。方法:将本实验室构建好的原核表达质粒pET28a(+)-FAPαc转化大肠杆菌BL21,IPTG诱导表达目的蛋白His-FAPαc;用纯化的目的蛋白免疫新西兰大白兔,获得兔抗小鼠FAPα多克隆抗体;用免疫印迹法和酶联免疫吸附试验检测该多克隆抗体与重组蛋白His-FAPαc的反应性及检测该多克隆抗体的效价,并用该抗体检测FAPα在肿瘤细胞和成纤维细胞以及肿瘤间质中的表达情况。结果:HisFAPαc融合蛋白可与兔抗小鼠FAPα多克隆抗体特异性结合;该多克隆抗体的效价高,特异性好,并且能检测小鼠胚胎成纤维细胞(Mouse embryo fibroblast cells;MEF)及结肠癌和乳腺癌的间质细胞高表达FAPα的全长序列蛋白。结论:制备的兔抗小鼠FAPα多克隆抗体能够有效地识别小鼠胚胎成纤维细胞和肿瘤间质细胞表达的FAPα蛋白,为研究FAPα在肿瘤的发生、发展及转移中的作用和以FAPα为靶点而开展的的抗肿瘤研究奠定基础。

Objective：To express and purify His-FAPαc fusion protein and to generate rabbit anti-mouse FAPα polyclonal antibody,which was used to analyze FAPα expression in tumor cells,Fibroblast cells and tumor stroma.Methods：A prokaryotic expression plasmid of pET28a（＋）-FAPαc constructed by our laboratory was transformed into BL21.The expression of His-FAPαc protein was induced by IPTG.The anti-mouse FAPα polyclonal antibody was obtained by immunizing rabbits with purified His-FAPαc protein.The quality of the antibody was identified by Western blot and ELISA.Whether FAPα expression in mouse tumor cells,fibroblast cells and tumor stroma was also analyzed using the antibody.Results：His-FAPαc fusion protein was specifically combined with rabbit anti-mouse FAPα polyclonal.The polyclonal antibody was of high titer with high specificity.It could recognize FAPα expression in mouse embryo fibroblast cells and tumor stroma.Conclusion：The rabbit anti-mouse FAPα polyclonal antibody could recognize FAPα expression in mouse embryo fibroblast cells and tumor stroma.Therefore,providing a tool to study the role of FAPα in tumor generation,promotion and metastasis.It also establishes a foundation of further anti-tumor research.