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目的:纯化人vasorin(VASN)蛋白胞外结构域的单克隆抗体并鉴定。方法:用表达人VASN蛋白胞外结构域单克隆抗体的杂交瘤细胞免疫小鼠,收集腹水,纯化抗体;ELISA检测单抗的特异性和亲和力,Western印迹、免疫共沉淀、细胞免疫荧光等实验检测单抗的特异性与可能的用途。结果:纯化获得2株抗VASN胞外结构域单抗V20和V21,ELISA结果显示二者均特异性强、亲和力高;Western印迹显示2株单抗均可结合Hep G2细胞中的VASN蛋白,以V20为佳;免疫共沉淀实验结果显示V21能够钓取Hep G2细胞中的VASN蛋白及细胞培养上清中的分泌型VASN;免疫荧光实验结果显示V21能与Hep G2细胞的VASN蛋白结合。结论:纯化获得2株抗人VASN胞外结构域单抗,为进一步研究VASN蛋白的生物学功能提供了实验工具。
OBJECTIVE: To purify and identify monoclonal antibodies against the extracellular domain of human vasorin (VASN) protein. Methods: The mice were immunized with the hybridoma cells expressing human extracellular domain of human VASN protein, the ascites was collected and the antibodies were purified. The specificity and affinity of McAbs were detected by ELISA, Western blotting, co-immunoprecipitation and immunofluorescence Detect the specificity and possible uses of mAbs. Results: Two anti-VASN anti-VASN extracellular domain monoclonal antibodies V20 and V21 were purified. The results of ELISA showed that the two antibodies were highly specific and had high affinity. Western blotting showed that both mAbs could bind VASN protein in Hep G2 cells, V20. Immunoprecipitation assay showed that V21 could secrete VASN protein in Hep G2 cells and secreted VASN in cell culture supernatant. Immunofluorescence assay showed that V21 could bind VASN protein in Hep G2 cells. CONCLUSION: Two anti-human VASN extracellular domain monoclonal antibodies were purified and provided an experimental tool for further study on the biological function of VASN protein.