【摘要】 目的表达和纯化CD112胞膜外区重组蛋白,制备针对CD112胞膜外区的单克隆抗体(mAb),并且研究CD112的分布。方法采用基因重组技术在真核系统表达CD112-Fc融合蛋白,亲和层析法进行蛋白纯化;纯化的CD112-Fc融合蛋白免疫BALB/c小鼠,杂交瘤技术建立分泌CD112mAb的杂交瘤细胞株,间接ELISA、Western blot及FCM鉴定mAb的特异性。用流式细胞术(FCM)检测CD112的细胞系分布。结果构建了高效真核表达载体pIg3C-CD112,表达并纯化了CD112-Fc融合蛋白,其纯度大于90%。以纯化重组蛋白为免疫原共制备9株分泌抗CD112mAb的杂交瘤细胞株(命名为FMU-CD112.1~FMU-CD112.9),制备腹水并纯化mAb。FMU-CD112.1、3、6和8能用于Western blot,FMU-CD112.1、4、6和8可用于FCM。CD112细胞分布检测显示该分子主要分布在正常上皮及内皮细胞系、巨核细胞谱系和部分T细胞系,并高表达于上皮及内皮来源的肿瘤、胶质瘤等细胞系。结论成功地表达纯化了CD112-Fc融合蛋白并建立了稳定分泌CD112mAb的杂交瘤细胞株,为CD112的功能研究打下坚实的基础。更多还原

【Abstract】 AIM: To express and purify fusion protein of human CD112 extracellular region, prepare monoclonal antibodies (mAb) to CD112 and investigate the distribution of CD112 molecule. METHODS: The CD112-Fc fusion protein was expressed with gene recombinant technique in eukaryotic system and purified with affinity chromatography column. The BALB/c mice were immunized with purified CD112-Fc for preparation of mAb by hybridoma technique. The prepared mAbs were identified by indirect ELISA, Western blot and flow cytometry (FCM). Subsequently, the distribution of CD112 on different human cell lines was investigated by FCM. RESULTS: The effective expression plasmid pIg3C-CD112 was constructed, and human CD112-Fc was successfully expressed and purified with a purity of more than 90%. Nine clones of hybridoma were obtained, which were designated as FMU-CD112.1-FMU-CD112.9. FMU-CD112.1, 3, 6 and 8 specifically reacted with recombinant CD112-Fc protein in Western blot and FMU-CD112.1, 4, 6 and 8 could be used in FCM. The investigation of CD112 distribution showed high expression on the cell lines differentiated from epithelial cells. CONCLUSION: The hybridomas secreting mAbs to human CD112 are established successfully, which may lay the foundation for further research on the biological function of CD112.更多还原