【摘要】 目的:制备VSTM抗体,鉴定其性能。方法:VSTM1有两种主要的剪接体VSTM-v1和VSTM-v2。前者为膜分子,后者为分泌蛋白,与前者相比后者仅缺少穿膜区。构建VSTM-v2的原核表达载体,诱导表达并纯化带两种不同标签的VSTM-v2重组蛋白,Trx-His-S-VSTM1-v2和GST-VSTM1-v2。用前者免疫家兔制备VSTM1抗血清,ELISA检测效价;后者偶联Sepharose 4B制备免疫亲和层析柱对抗血清进行纯化。通过Western blot法和免疫荧光细胞化学技术,鉴定该抗体的特异性与适用范围。结果:免疫得到兔抗人VSTM1抗血清,效价达到1∶106。纯化后的抗体用于Western blot可特异识别过表达和内源性的VSTM1,并可用于免疫荧光细胞化学技术检测细胞膜表面过表达的VSTM-v1。结论:成功制备了兔抗人VSTM1抗体,具有较高的效价及特异性,可以识别内源性VSTM1并可用于免疫荧光细胞化学技术。更多还原

【Abstract】 AIM: To prepare and characterize the polyclonal antibody against human VSTM1.METHODS: VSTM1 has two main isoforms,VSTM1-v1,a type I transmembrane protein,and VSTM1-v2,a classical secretory protein,lacking only the transmembrane domain compared with VSTM1-v1.Two recombinant prokaryotic proteins of VSTM1-v2,Trx-His-S-VSTM1-v2 and GST-VSTM1-v2,were constructed,expressed,purified,and then used for immunization of New Zealand rabbits to prepare anti-VSTM1 antibody and coupling with CNBr-activated Sepharose 4B to purify the antibody by immunoaffinity chromatography,respectively.ELISA was performed to detect the titers of the antiserums.After purification,the antibody was identified by Western blotting and immunofluorescence cytochemistry.RESULTS: The titers of the antiserums from the two immunized rabbits were both 1∶ 106.Western blotting confirmed that the purified antibody could recognize both the overexpressed and endogenous VSTM1 specifically.Immunofluorescence cytochemistry verified that the antibody could also recognize the overexpressed VSTM1-v1 on the surface of HEK293T cells.CONCLUSION: The rabbit antibody against human VSTM1 of a high titer has been obtained,which can be used for recognizing endogenous VSTM1 in immunofluorescence cytochemistry.更多还原