凡纳滨对虾血清中直接与病原菌相结合的主要蛋白的鉴定

Identification of the main proteins binding with pathogen directly in Litopenaeus vannamei serum

以凡纳滨对虾为研究对象，将其血清分别与溶藻酸弧菌，哈维氏弧菌，嗜水气单胞菌和金黄色葡萄球菌等4种病原菌孵育，采用亲和蛋白质组学和Western-blotting等分子生物学方法纯化、鉴定和确证其中可与病原菌直接结合的蛋白。结果发现，与对照组相比4种病原菌与虾血清孵育5h后均可与对虾血清中分子质量约为75000（p75）的蛋白相结合，而孵育15h后既可与p75相结合，还可与分子质量约为77000（p77）的蛋白以及0～4种不同分子质量的蛋白相结合。经MALDI-TOF／MS分析和Mascot搜索引擎检索，p75和p77蛋白分别与凡纳滨对虾中分子质量约为75000、77000的2个血蓝蛋白亚基具有显著一致性。尤其是p75蛋白还可与兔抗血蓝蛋白75000亚基抗体发生特异性结合。由此推断，凡纳滨对虾血清中与病原菌直接结合的主要蛋白为血蓝蛋白，提示血蓝蛋白全蛋白可能具有抗菌活性。

It has been proved that natural immunity played an vital role in invertebrate animals, and mainly depended on non-specific immunity molecules with the mode of pathogen-associated molecular pattern to recognize pathogen and then to perform immune functions. In order to search the main proteins with function of recognizing directly pathogen in shrimp serum, an attempt was made to purify and identify the proteins bound directly to pathogens including Vibrio alginolyticus, Vibrio harveyi, Aeromonas hydrophila and Staphylococcus aureus in shrimp Litopertaeus vannamei serum using the methods of affinity proteornic and Western-blotting in the current study. The results showed that a protein with 75 000 （namely p75） in the shrimp serum could bind directly to the four pathogens in comparison with its control group respectively after the four types of pathogen reacted with shrimp serum for 5 h. While they reacted for 15 h another shrimp serum protein with 77 000 （namely p77） and several other proteins （0-4） with different molecular weights also bound directly to the four pathogens. Then p75 and p77 proteins were cut out from the gels and subjected to MALDI- TOF/MS analysis. The search results obtained from Mascot search engine in NCBInr database indicated that p75 and p77 proteins were identified to be Litopenaeus vannimei hemocyanin subunits with about 75 000 and 77 000 respectively. Moreover, the p75 protein was documented to be strongly reacted with rabbit anti-shrimp hemocyanin 75 000 subunit antibody specially. Thus, these results led to be deduced that hemocyanin was the main protein binding directly to different pathogens in the shrimp L. vannimei serum, suggesting that hemocyanin probably possesses directly antibacterial function and would be helpful for research of hemocyanin＇ s non-specific immune activities and its mechanism.