Department of Animal Health and Bioscience, Faculty of Agricultural Sciences, Aarhus University, 8830 Tjele, Denmark.
J Immunol Methods. 2010 Oct 31;362(1-2):204-8. doi: 10.1016/j.jim.2010.09.010. Epub 2010 Sep 9.
Bovine conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step procedure for the purification of conglutinin. In the first step, bovine serum is incubated with non-coupled chromatographic TSK beads at 37°C to allow complement activation and iC3b deposition on the beads and subsequent binding of conglutinin to iC3b. Conglutinin is then eluted from the beads by EDTA. In the second step, conglutinin is separated from iC3b and IgM by ion-exchange chromatography. This purification procedure yielded 81 μg of conglutinin per ml of serum with a recovery of 61.2%. Surface plasmon resonance analysis showed that the purified conglutinin had a high affinity for mannan (K(d)=2.3-3.2 nM). SDS-PAGE and time-resolved immunofluorometric assays showed that the conglutinin was not contaminated with other serum collectins such as collectin-43 or mannan-binding lectin.
牛血纤维蛋白原是一种参与固有免疫的血清蛋白。它依赖于钙离子与 iC3b 结合，iC3b 是补体成分 C3 在补体激活后沉积在细胞表面、免疫复合物或人工表面上的产物。我们在此提出了一种简单而有效的两步法来纯化血纤维蛋白原。在第一步中，牛血清在 37°C 下与非偶联的 TSK 珠孵育，以允许补体激活和 iC3b 沉积在珠上，随后血纤维蛋白原与 iC3b 结合。然后，EDTA 将血纤维蛋白原从珠上洗脱下来。在第二步中，通过离子交换层析将血纤维蛋白原从 iC3b 和 IgM 中分离出来。该纯化程序每毫升血清可获得 81μg 的血纤维蛋白原，回收率为 61.2%。表面等离子体共振分析表明，纯化的血纤维蛋白原对甘露聚糖具有高亲和力（K(d)=2.3-3.2 nM）。SDS-PAGE 和时间分辨免疫荧光测定表明，血纤维蛋白原没有被其他血清凝集素如凝集素-43 或甘露聚糖结合凝集素污染。
