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目的 检测人和兔中性粒细胞防御素的抗SARS冠状病毒活性 ,探讨防御素在SARS发生发展过程中的作用。方法 应用酸 尿素 聚丙烯酰胺凝胶洗脱电泳(CAU PAGE)从人和兔中性粒细胞中分别纯化人中性粒细胞防御素 (HNP1 3)和兔中性粒细胞防御素(RNP1 5 ) ,通过微量培养法观察不同浓度防御素对SARS冠状病毒BJ0 1株感染Vero E6细胞及病毒复制的影响。结果 用CAU PAGE分离的HNP1 3和RNP1 5经 15 %SDS PAGE电泳后在分子量 4 0 0 0左右呈现单一条带 ,HNP1 3经ELISA定量检测确认。HNP1 3和RNP1 5的抗大肠杆菌 (ATCC2 5 92 2菌株 )活性的IC50 分别在 2 5～ 5 0 μg/ml和 5～ 10 μg/ml之间 ,保持了天然的抗菌活性。在抗SARS冠状病毒实验中 ,HNP1 3和RNP1 5在 2 5～10 0 μg/ml浓度时 ,不能阻断SARS冠状病毒致细胞病变效应 (CPE) ,也不能抑制SARS冠状病毒在Vero E6细胞中的复制。结论 天然防御素HNP1 3和RNP1 5对SARS冠状病毒没有抑制和杀灭活性 ,可能提示家兔和人对SARS冠状病毒不具备天然免疫力
Objective To detect the anti-SARS coronavirus activity of human and rabbit neutrophil defensins and to explore the role of defensins in the development of SARS. Methods Human neutrophil defensin (HNP1 3) and rabbit neutrophil defensin (RNP1 5) were purified from human and rabbit neutrophils by aciduric acid polyacrylamide gel electrophoresis (CAU PAGE) The effect of different concentrations of defensins on the replication of Vero E6 cells and virus in SARS-Covirus BJ0 1 strain was observed by micro culture method. Results HNP1 3 and RNP1 5 separated by CAU PAGE showed a single band on the molecular weight of 40,000 after electrophoresis with 15% SDS PAGE. HNP1 3 was confirmed by ELISA. The IC50 values of HNP1 3 and RNP1 5 against Escherichia coli (ATCC2 5 92 2) ranged from 25 μg / ml to 50 μg / ml and from 5 μg / ml to 10 μg / ml, respectively, and maintained their natural antibacterial activity. In anti-SARS coronavirus experiments, HNP1 3 and RNP1 5 could not block the cytopathic effect (CPE) of SARS coronavirus or inhibit the expression of SARS-CoV in Vero E6 cells at the concentration of 25 ~ 100 μg / ml Copy. Conclusion The natural defensins HNP1 3 and RNP1 5 have no inhibitory and killing activity against SARS coronavirus and may suggest that rabbits and humans do not possess the natural immunity against SARS coronavirus