红芸豆红细胞凝集素单体分离纯化及性质探究.docVIP

红芸豆红细胞凝集素单体分离纯化及性质探究 　【摘要】 目的 研究一种从红芸豆中提取红细胞凝集素单体(PHA-E)的新方法。方法 红芸豆经过浸提，离子交换树脂分离，分子筛层析纯化后得到PHA-E样品。采用电泳法测定其纯度、分子量和等电点。用2%人细胞悬液测定样品凝集红细胞的能力和影响凝血的因素，使用硫酸苯酚法测定其糖含量。结果 经PAGE分析PHA-E样品为单带电泳纯，SDS上显示亚基分子量为32 kD，等电点为6.5.样品使人红细胞50%凝集的蛋白质最低浓度为4 μg/ml左右。单糖不影响PHA-E凝血活力，EDTA抑制其凝血活力，Zn2+促进其凝血。糖含量为8.1%. 【关键词】 红细胞凝集素 分离纯化 凝血 性质研究 Purification and Partial Characterization of Erythrohemagglutinin from Phaseolus Vulgaris HE Tao1,ZHANG Tao2*,WU Chang-ying1,WANG Liang1,SHAN Xiao-xue1,LUO Qin1 (School of Preclinical Medicine,Chengdu Medical College,1.2006s Biotechnology,2.Biotechnology laboratory,Chengdu,610083,China) Abstract：Objective To study a new method for Erythrohemagglutinin（PHA-E）production from Phaseolus vulgaris.Methods PHA-E was purified from the excellent seeds of Phaseolus vulgaris by extraction，ion exchange chromatography and final gel filtration chromatography.The purity,the molecular weight and the isoelectric point of purified PHA-E were determined by electrophoresis.The ability and the influencing factor of agglutination were determined by 2% erythrocytes of human.Conclusion The purified PHA-E was single PAGE outline and had apparent subunit molecular weights of 32 kD by SDS-polyacrylamide gel electrophoresis.Isoelectric point of the PHA-E was 6.5.PHA-E can promote agglutination of human erythrocytes at 4 μg/ml,but its activity was not inhibited by any monosaccharide.EDTA can inhibit the ability of agglutination,and Zn2+ can accelerate it.The sugar content of PHA-E was 8.1%. Key words:erythrohemagglutinin；separation and purification；haemagglutination；characterization 植物凝集素(Phytohemagglutinin PHA)是从豆科植物种子中提取的一种含糖蛋白质［1］。植物凝集素具有刺激外周血淋巴细胞RNA合成和有丝分裂［2］，促进免疫反应，使细胞凝聚，肿瘤细胞失活，以及诱发人外周血淋巴细胞产生抗癌淋巴因子等生物学活性。文献［3］报道PHA是由E、L两种亚基组成的四聚体蛋白质的混合物，按其亚基组成可分为L4、L3E、L2E2、LE3、E4具有相同物化性质和不同生物学活性的同工凝集素。其中PHA-E含有4个相同的E亚基，具有最强的红细胞凝集功能，没有白细胞凝集能力。由于凝集素单体较混合物PHA对于糖链具有更加特异的识别能力，可以作为分子探针，研究特异细胞膜受体的分布等，在免疫学、肿瘤、生殖生理、细胞生物学等许多方面得到应用，在医学、农业上呈现出巨大的应用前景。关于红细胞凝集素单体的分离纯化国内还未见报道，本研究改进和优化前人的提取和纯化方法，简化操作程序，从红