目的 对黑尾胡蜂磷脂酶的分离方法和活性进行研究，为黑尾胡蜂的毒性机制研究和药用发掘提供实验数据。方法 通过葡聚糖凝胶G-75分子筛柱和肝素亲和柱对黑尾胡蜂毒液中的磷脂酶活性成分进行分离。利用质谱对目的蛋白进行肽指纹图谱分析，利用蛋白测序仪测定目的蛋白N末端氨基酸序列，从结构上对目的蛋白进行鉴定。利用磷脂酶A1检测试剂盒确认目的蛋白对底物磷脂的特异性水解位点，从活性上对目的蛋白进行鉴定。通过血浆复钙时间的测定检测目的蛋白对凝血系统的影响。结果 通过葡聚糖凝胶G-75分子筛柱和肝素亲和柱两步分离纯化，成功从黑尾胡蜂蜂毒中纯化到了1个磷脂酶活性组分。该组分在SDS-PAGE凝胶中表观相对分子质量约为32 000，因此命名为Vtp32。肽指纹图谱分析及N端氨基酸序列比对结果显示Vtp32与Vespa属胡蜂磷脂酶A1（phospholipase A1，PLA1）具有高度的序列相似性。Vtp32可以特异性水解磷脂的sn-1位，因此Vtp32为黑尾胡蜂PLA1。Vtp32水解磷脂可产生溶血性凝脂，导致红细胞裂解。Vtp32可显著延长人血浆的复钙时间，具有抗凝活性。结论 黑尾胡蜂毒液中含有大量的PLA1，可通过葡聚糖凝胶G-75分子筛柱和肝素亲和柱两步分离方法进行纯化。黑尾胡蜂PLA1具有溶血和抗凝活性。

Objective To find an effective method to isolated phospholipase from Vespa tropica ducalis and characterize its biological activities to support the pathogenic mechanism research and officinal value exploitation in the future. Methods The component with phospholipase activity was isolated by successive gel filtration (Sephadex G-75, supferfine) and heparin affinity chromatography steps (Hitrap Heparin HP). The protein was identified by peptide mass fingerprinting, N-terminal amino acid determination and blast analysis, as well as phospholipase A1 (PLA1) activity monitor. Plasma recalification time test was employed to detect the effect of Vtp32 on coagulation. Results A protein with phospholipase activity was orderly separated and purified from V. tropica ducalis venom using gel filtration and heparin affinity chromatography. The purified protein was homogenous on the SDS-PAGE gel with relative molecular mass of 32 000, so it was termed as Vtp32. Peptide mass fingerprinting assay and N-terminal amino acid sequence blast result revealed that Vtp32 showed high homologous with PLA1 from wasp of Vespa genus. In addition, Vtp32 hydrolyzed the sn-1 ester linkage of phospholipids. These results indicated that Vtp32 was PLA1 from V. tropica ducalis. Vtp32 hydrolyzed phosphatidylcholine, and the hydrolysis product can lyse human erythrocytes. Vtp32 delayed the recalification time of human plasma and hence had anti-coagulation activity. Conclusion PLA1 is widely existed in the venom from V. tropica ducalis. Gel filtration followed by heparin affinity chromatography is an effective isolation strategy for the purification of PLA1. The results show that V. tropica ducalis PLA1 has hemolytic and anticoagulative activity.

国家自然科学基金资助项目（81373945，31460571）