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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。
Objective: To prepare VSTM antibody and identify its properties. Methods: VSTM1 has two major splice variants VSTM-v1 and VSTM-v2. The former is a membrane molecule, the latter is a secreted protein, compared with the former only lacking the latter. The prokaryotic expression vector of VSTM-v2 was constructed to express and purify VSTM-v2 recombinant protein, Trx-His-S-VSTM1-v2 and GST-VSTM1-v2 with two different tags. VSTM1 antiserum was prepared by immunizing rabbits with the former and the titer was detected by ELISA. The latter was coupled to Sepharose 4B to prepare immunoaffinity chromatography column to purify the antisera. Western blot and immunofluorescence cytochemistry were used to identify the specificity and applicability of the antibody. RESULTS: Rabbit anti-human VSTM1 antiserum was obtained and the titer reached 1:106. The purified antibody can be used to detect the over-expressed and endogenous VSTM1 specifically by Western blot and can be used to detect the over-expressed VSTM-v1 on the cell surface by immunofluorescence cytochemistry. CONCLUSION: The rabbit anti-human VSTM1 antibody has been successfully prepared and has high titer and specificity. It can recognize endogenous VSTM1 and can be used in immunofluorescence cytochemistry.