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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%。
The cytoplasmic granules were isolated from human neutrophils and the acid-soluble fractions were extracted from the medium with 5% acetic acid. Purified human neutrophil defensin (HNP) was obtained by gel filtration chromatography on Sephadex G100 and Bio-gel P10. Purified HNP was analyzed by AU-PAGE for a band containing no foreign protein and was analyzed by SDS-PAGE as a band with a molecular weight of about 3,000. Amino acid composition analysis showed that among the 17 kinds of amino acids tested, HNP contained 12 kinds, and the deduced amino acid residues of all kinds of amino acid residues were consistent with the amino acid sequence analysis results reported in the literature. HNP showed strong Candida albicans activity in vitro, and the mortality of Candida albicans was nearly 90% at the concentration of 100μg / ml.