以凡纳滨对虾(Litopenaeus vannamei)为研究对象,通过分子筛层析、Tricine-SDS-PAGE、Western-blotting、凝集实验、抑菌实验和Edman N端测序等方法探索血蓝蛋白酶解多肽的凝集和抑菌活性.结果发现,血蓝蛋白经胰蛋白酶酶解后可产生分子量约为6—70 kD的7条多肽,该酶解混合物对副溶血弧菌(Vibrio parahaemolyticus)具有显著的凝集活性,与未酶解的血蓝蛋白相比,其凝集活性可提高4—16倍.在此基础之上,进一步分离纯化该7条多肽,发现多肽-3对副溶血弧菌表现出较强的抑菌活性,且具有较好的浓度依赖性.在浓度为75μg/mL时,其抑菌率为(93.76±1.60)％,与阴性对照组相比,存在极显著性差异(P<0.01).进一步研究显示该多肽位于血蓝蛋白N端α-螺旋区域.由此推测,凡纳滨对虾血蓝蛋白在体外经胰蛋白酶酶解后可产生具有凝集、抑菌等免疫活性的多肽,这对研究血蓝蛋白的降解机制及其在先天免疫中的作用具有重要意义.%Previous studies have demonstrated antibacterial and antifungal activities of hemolymph-derived hemocyan-in upon pathogen infection with unknown mechanisms. To investigate how peptides derive from hemocyanin, multiple methods including size-exclusion chromatography, Tricine-SDS-PAGE, immunoblotting, bacterial agglutinative and antibacterial assays were applied. The results showed that shrimp Litopenaeus vannamei hemocyanin could produce seven peptides via trypsin digestion, ranging from 6 to 70 kD, which could be identified by rabbit anti-shrimp hemoc-yanin antibody specifically. These peptides aggregated with Vibrio parahaemolyticus in vitro, whose agglutinative acti-vity was 4—16 folds higher than that of full length hemocyanin. The peptide 3 possessed obvious antibacterial activit-ies against V. parahaemolyticus with a antibacterial rate (93.76±1.60)％ at the concentration of 75 μg/mL, which was significantly higher than that of the control group (P<0.01). In addition, N-terminal Edman Sequencing analysis showed that the peptide 3 was located in the α-helix region of N-terminus of shrimp L. vannamei hemocyanin small subunit. These discoveries will help to understand how the hemocyanin derived peptides are formed and to establish effective strategies for shrimp disease control.
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