凡纳滨对虾血蓝蛋白与病原菌凝集作用靶标的鉴定

血蓝蛋白是一种具有多种非特异性免疫学活性的多功能蛋白，以前的研究发现,血蓝蛋白具有凝集活性.本研究采用凝集抑制实验和亲和蛋白质组学等方法探索凡纳滨对虾血蓝蛋白与病原菌的凝集作用靶标.结果显示，大肠杆菌K12和副溶血弧菌外膜蛋白可以抑制血蓝蛋白对7种细菌的凝集活性，其中大肠杆菌K12中2种分子质量分别为16 kD、18 kD （命名为 p16、p18）的外膜蛋白可以与血蓝蛋白发生特异性的结合，经MALDI-TOF/MS鉴定，p16、p18 分别与大肠杆菌外膜蛋白OmpC、OmpX具有高度同源性.尤其是与大肠杆菌K12野生菌株相比，血蓝蛋白对 ΔOmpX 的凝集特异性明显降低，后者仅为前者的25％.由此推测，OmpX 应为血蓝蛋白与病原菌的凝集作用靶标.

Identification of the Pathogen Agglutinative Target Recognized by Litopenaeus vannamei Hemocyanin

Hemocyanin is a non-specific immune protein with multi-functions, and its agglutinative activity has previous reported from our work. In this study, we used agglutination inhibition assay and affinity proteomic technology to further investigate the agglutinative target of hemocyanin. The results have shown that the outer membrane proteins (Omps) of Escherichia coli K12 and Vibrio parahaemolyticus could completely inhibit the agglutinative activity of Litopenaeus vannamei hemocyanin against seven bacterial species, including E.coli K12, V. parahaemolyticus, Vibro alginolyticus, Vibro fluvialis, Vibro harveyi, Aeromonas hydrophila and Staphylococcus aureus. Two specific Omps with molecular weight of 16 kD and 18 kD (p16 and p18) identified by MALDI-TOF/MS from E. coli K12 were able to bind hemocyanin, which were homologous with OmpC and OmpX of E.coli, respectively. The specific agglutinative activity of hemocyanin with △OmpX E. coli was substentially decreased by 75% as comparison with wild type K12. We conclude that OmpX appeared to be the pathogen agglutinative target that recognized by hemocyanin.