与不同病原菌相结合的凡纳滨对虾血蓝蛋白溶血活性对比分析

摘要

In this study, we investigated the diversities of six Litopenaeus vannamei hemocyanin isomers binding to dif-ferent bacteria. The methods of affinity-binding, PAGE, SDS-PAGE, Western-blotting and hemolytic activity assays were used. The results indicated that all the six hemocyanin isomers, namely hemocyanin isomer directly binding to Vibrio parahaemolyticus (HMC-VP), Vibrio alginolyticus (HMC-VA), Vibrio fluvialis (HMC-VF), Escherichia coli K12 (HMC-EC), Beta Streptococcus (HMC-BS) and Staphylococcus aureus (HMC-SA) displayed different hemolytic activities to chicken erythrocyte. Among of these, HMC-VP and HMC-SA showed the highest hemolytic activity and reached almost complete (100.00%), while that of HMC-VA was only 39.96%. To further elucidate the molecular basis underlying hemocyanin isomers functional diversities, the assays of glycosyl-oxidation and trypsin digestion were selected. The results showed that glycosyl-oxidation led to a generally significant decrease in hemolytic activity of all of the hemocyanin iso-mers, even completely abolished. In contrast, the hemolytic activities of these hemocyanin isomers were highly increased, even reached 100.00%after trypsin digestion. Thus, these results revealed that six hemocyanin isomers possessed different hemolytic activities, which may be related to the diversity of protein glycosylation and conformation.%以凡纳滨对虾(Litopenaeus vannamei)为研究对象，采用亲和孵育、PAGE、SDS-PAGE、Western-blotting、溶血活性测定等技术，探索与6种不同病原菌相结合的血蓝蛋白溶血活性的差异。结果发现，与副溶血弧菌(Vibrio parahaemolyticus)、溶藻酸弧菌(Vibrio alginolyticus)、河弧菌(Vibrio flurialis)、大肠杆菌(Escherichia coli K12)、乙型链球菌(Beta Streptococcus)和金黄色葡萄球菌(Staphylococcus aureus)6种不同细菌相结合的血蓝蛋白(分别命名为 HMC-VP、HMC-VA、HMC-VF、HMC-EC、HMC-BS、HMC-SA)对鸡红细胞表现出不同的溶血活性，其中HMC-VP、HMC-SA溶血活性最高(100.00%)， HMC-VA溶血活性最低(39.68%)。在此基础之上，进一步采用糖基氧化和胰蛋白酶消化等策略探索引起该6种血蓝蛋白溶血活性差异的分子基础。结果表明，该6种血蓝蛋白经糖基氧化后，溶血活性大幅度下降抑或丧失，而经胰蛋白酶水解后，溶血活性大幅度升高抑或达到100.00%。由此说明，与不同病原菌相结合的血蓝蛋白免疫学功能(溶血活性)存在显著性差异，造成该差异的原因可能与血蓝蛋白的糖基化修饰、蛋白构象的多样性有关。