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为鉴定猪肾上腺皮质细胞膜是否存在高密度脂蛋白结合蛋白,并研究其特性,从新鲜猪肾上腺皮质细胞膜提取高密度脂蛋白结合蛋白,应用亲和层析法将其初步纯化。获得的可溶性蛋白经Ligandblot结合分析和Dotblot法测定,结果发现此膜蛋白与高密度脂蛋白有高度亲和力,用Westernblot法测得该蛋白的分子量为80kDa,亲和层析提取得到的结合蛋白分子量为77kDa。应用生物素-亲和素酶联免疫吸附试验测定该蛋白的结合特性:平衡解高常数(kd)=21.6μg/g,最大结合量(Bmax)=678.2mg/L蛋白。结果提示纯化的猪肾上腺皮质细胞膜高密度脂蛋白结合蛋白具有很高的亲和力,证明亲和层析法条件温和。
To identify the existence of high density lipoprotein-binding protein in porcine adrenocortical cell membrane and study its characteristics, high density lipoprotein-binding protein was extracted from fresh porcine adrenocortical cell membrane and purified by affinity chromatography. The soluble protein obtained by Ligandblot binding assay and Dotblot assay showed that the membrane protein and high density lipoprotein has a high affinity, using Westernblot method to determine the molecular weight of 80kDa, affinity chromatography extraction of the resulting binding protein molecular weight 77kDa. The biotin-avidin-linked immunosorbent assay was used to determine the binding properties of the protein: kd = 21.6μg / g, Bmax = 678.2mg / L protein. The results suggest that purified porcine adrenocortical membrane HDL-binding protein has a high affinity and proves that the conditions of affinity chromatography are mild.