摘要
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 obtain monoclonal antibodies（m Ab） against vasorin ectodomain and investigate the appli-cations of these antibodies. Methods： Antibodies were purified from ascites fluid of mice, which were immunizedwith hybridoma colonies. ELISA was used to examine specificity and relative affinity of the antibodies. Westernblot assay, immunoprecipitation and immunofluorescence were used to identify the antibodies＇ specificity and appli-cations. Results： The results of ELISA showed that two anti-vasorin ectodomain m Abs were with high specificityand relative affinity. Both V20 and V21 could bind vasorin in the cell lysates from Hep G2 cell in Western blotassay, and V20 was much better. V21 could bind vasorin in Hep G2 cell and Hep G2 medium in immunoprecipita-tioin assay. Moreover, V21 could also bind vasorin of Hep G2 in immunofluorescence assay. Conclusion： The anti-vasorin ectodomain m Abs with high affinity and specificity provide a powerful experimental tool to further addressthe biological functions of vasorin.
出处
《生物技术通讯》
CAS
2016年第2期216-218,共3页
Letters in Biotechnology
基金
国家自然科学基金(81572846)
