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目的建立适用于复杂体系的神经氨酸酶抑制剂毛细管电泳筛选方法。方法以神经氨酸酶为靶点,以2′-(4-甲基伞形酮-α-N-乙酰神经氨酸)为底物,建立神经氨酸酶抑制剂的毛细管电泳筛选方法,并将该方法用于海洋药物可口革囊星虫粗提物的活性筛选。结果以10mmol·L-1pH=10.0的硼砂为背景电解质,酶促反应底物与产物在4.5min内基线分离。酶促反应在含4mmol·L-1 CaCl2的pH=3.5的磷酸体系中,37℃水浴中酶与底物孵育15min,产物有稳定的较大的生成量。扎那米韦阳性对照验证结果表明,该方法可用于神经氨酸酶抑制剂筛选。对源于可口革囊星虫的11个粗提物筛选结果显示,7个粗提物对神经氨酸酶有抑制活性。结论系统优化了毛细管电泳分离分析方法及神经氨酸酶反应体系,建立的模型可用于复杂体系中抗流感病毒药物神经氨酸酶抑制剂的筛选。
Objective To establish a neuramidase inhibitor capillary electrophoresis screening method suitable for complex systems. Methods Neuraminidase was used as the target and 2 ’- (4-methylumbelliferone-α-N-acetylneuraminic acid) was used as the substrate to establish the neuramidinase inhibitor capillary electrophoresis screening method. The method was applied to the active screening of the crude extract of marine drug. Results Borax with 10 mmol·L-1 pH = 10.0 was used as the background electrolyte, and the substrate and product of the enzymatic reaction were separated within 4.5 min. Enzymatic reaction in the pH = 3.5 containing 4mmol·L-1 CaCl2 phosphate system, 37 ℃ water bath enzyme incubated with the substrate for 15min, the product has a stable and large formation. Zanamivir positive control validation results show that this method can be used for neuraminidase inhibitor screening. The results of 11 crude extracts from the protozoal cyst Nematode showed that seven crude extracts had inhibitory activity on neuraminidase. Conclusion The capillary electrophoresis separation and analysis system and neuraminidase reaction system are optimized systematically. The established model can be used to screen anti-influenza virus neuraminidase inhibitors in complex system.