凡纳滨对虾血蓝蛋白的细菌凝集活性

Bacterial agglutinative activity of hemocyanin in shrimp Litopenaeus vannamei

以凡纳滨对虾（Litopenaeaus vannarnei）为研究对象，运用亲和层析、PAGE、SDS-PAGE、Western-blotting和细菌凝集实验等方法探索凡纳滨对虾血蓝蛋白的细菌凝集活性。结果发现，血蓝蛋白对副溶血弧菌（Vibrio parahaemolyticus）、溶藻酸弧菌（Vibrio alginolyticus）、河弧菌（Vibrio fluvialis）、哈维氏弧菌（Vibrio harveyi）、嗜水气单胞菌（Agromona hydrophila）和金黄色葡萄球菌（Stphylococcus aureus）等6种虾类致病菌具有凝集活性，其凝集活性大小为0．27-1．08mg／L，其中对副溶血弧菌和溶藻酸弧菌凝集活性最大，是哈维氏弧菌的4倍。在此基础之上，选取α-D-葡萄糖、D-半乳糖、D-木糖、D-山梨糖、蔗糖、麦芽糖、α-D-乳糖、甘露醇和N-乙酰神经氨酸等9种糖考察其对血蓝蛋白细菌凝集活性的影响。结果显示，血蓝蛋白的细菌凝集活性可被α-D-葡萄糖和N-乙酰神经氨酸所部分抑制和全部抑制，其最低抑制浓度范围为50～100mmol／L。由此推测，血蓝蛋白确实具有细菌凝集活性，这对进一步研究血蓝蛋白的抗菌机理具有重要意义。

Hemocyanin is a kind of important multifunctional protein found in the hemolymph of both arthropod and mollusk, although this copper-containing pigment is generally accepted as an oxygen carrier. Particularly, recent reports have demonstrated that hemocyanin was probably an important type of defense molecule, which not only could be functionally converted into a phenoloxidase-like enzyme by some reagents,but also could produce C-terminal fragments with broad antifungal and antibacterial activities. However, up to date no research about hemocyanin＇ s bacterial agglutinative activity was reported. Thus, in the paper, an attempt was made to testify whether Litopenaeus vannamei hemocyanin possesses bacterial agglutinative function. The methods of affinity chromatography, PAGE, SDS-PAGE, Western-blotting, bacterial agglutinative test and saccharide agglutinating-inhibition test were used. In the experiment, six kinds of bacteria including Vibrio parahaemolyticus , V. alginolyticus , V. fluvialis , V. harveyi , Aerornona hydrophila and Stphylococcu aureus were selected to react with Litopenaeus vannamei hemocyanin. The resuits showed that hemocyanin could aggregate with the six types of bacteria respectively, whose agglutinative activity was from 0.27 to 1.08 mg/L. Then saccharide specificity of hemocyanin was analyzed by nine types of saccharide including α-D-Glucose, D-Galactose, D-Xylose, D-Sorbose, Sucrose, Maltose, α-D-Lactose, Mannitol and N-Acetylneuraminic acid. The results indicated that the agglutinative activity of hemocyanin could be partly inhibited by α-D-Glucose at the concentration of 200 mmol/L in the presence of S. aureus, and completely inhibited by N-Acetylneuraminic acid, whose minimum concentration was from 50 to 100 mmol/L,in the presence of the six types of bacteria respectively. But the results can＇t be obtained by the other saccharides, even at the concentration of 200 mmol/L. Thus, these results indicate that Litopenaeus vannamei hemocyanin indeed possesses bacterial agglutinative function, which is the newest discovery in the field of immune function of hemocyanin and will be helpful for its further research and practical application.