人中性粒细胞防御素的分离纯化

Purification and Charaterization of Human Neutrophil Defensins

从人中性粒细胞分离胞浆颗粒,以5%乙酸从中提取酸溶性组分,经SephadexG100及Bio-gel P10凝胶过滤层析后获得纯化的人中性粒细胞防御素(HNP)。纯化的HNP经AU-PAGE分析不含杂质蛋白区带,SDS—PAGE分析为一条分子量约3 000的带。氨基酸组成分析表明,在被检测的17种氨基酸中,HNP含12种,各种氨基酸残基的推算数与文献报道氨基酸序列分析结果一致。HNP在体外实验中表现较强杀白色念珠菌活性,在浓度为100μg/ml时,白色珠菌死亡率近90%。

Defensins, a family of 3-4 KD cationic peptides, have been found recently in the cytoplasmicgranules of human, rabbit, guinea pig and rat polymorphonuclear neutrophils and rabbit alveolarmacrophages. The defence roles of these molecules, such as antimicrobial activity, viral inactiva-tion, tumor cell cytotoxicity, chemotactic and opsonic actions, etc. have been adequately studied.In order to investgate the role of Defensins in the neutrphil-mediated pathological process ofsome diseases, we isolated and purified human neutrophil Defensins. By using Sephadex G100and Bio-gel P10 gel filtration chromatography, the purified molecules were obtained. The puritywas identified by AU-PAGE,SDS-PAGE, and amino acid composition analysis. In vitro fugicidalactivity of the purified human neutrophil Defensins against Candida albicans was also examined.When incubated with 33 Mol concentration of human Defensins, more than 90% of Candida,al-bicans were killed in the methylene blue assay system.