与凡纳滨对虾抗副溶血弧菌有关的6.0kD血蓝蛋白降解肽段 [中文引用][英文引用]

以凡纳滨对虾(Litopenaeus vannamei)为研究对象,采用人工感染、Tricine-SDS-PAGE、Western-blotting、MALDI-TOF/TOF、抑菌实验等方法对血蓝蛋白小分子降解肽段进行研究。结果发现,副溶血弧菌(Vibrio parahaemolyticus)刺激对虾24 h后,与对照组相比,其血淋巴中新出现5个分子量为6.0~31.0 kD的与兔抗血蓝蛋白抗体呈阳性的小分子条带。其中,分子量为6.0 kD的a条带(命名为HMCp6肽段)与凡纳滨对虾血蓝蛋白具有高度同源性,对副溶血弧菌具有明显的抑菌活性,与对照组相比,存在极显著性差异(P < 0.01)。由此说明, HMCp6应该是对虾感染副溶血弧菌后所产生的一种新的小分子降解肽段,推测其在对虾抗感染防御中发挥重要作用。

Recent reports have revealed that hemocyanin in shrimp and other invertebrates is a novel multifunctional protein with immunological activities, such as phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. Moreover, several studies-including our own previous findings-indicate that hemocyanin may produce some functional fragments involved in pathogen resistance in shrimp. However, no study has comprehensively evaluated these fragments. We conducted a comparative analysis in the proteome of the shrimp Litopenaeus vannamei after the shrimp were infected with Vibrio parahaemolyticus for 24 h to search for novel hemocyanin fragments. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses of hemolymph drawn directly from the pericardial sinus revealed five peptide fragments (bands a, c, e, f, g), ranging from 6.0 to 31.0 kD, that bound to anti-shrimp hemocyanin antibodies in plasma, compared with those in the control group, suggesting that they might be hemocyanin degradation products. Moreover, a 6.0 kD peptide fragment (named HMCp6) was subjected to matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry analysis, and the database search results indicated that the HMCp6 peptide fragment was highly homologous with two subunits of L. vannamei hemocyanin (gi|325462537 and gi|854403). This result is similar to the finding that the C- and N-terminal hemocyanin fragments are significantly altered in Penaeus vannamei hemocytes during Taura syndrome virus infection. The HMCp6 peptide fragment possessed clear antibacterial activities (antibacterial rate, 98%) against V. parahaemolyticus. This result agrees with a previous finding that a 1.9 kD antibacterial peptide from Pacifastacus leniusculus hemocyanin possesses clear antibacterial activities against Gram-positive bacteria. In conclusion, our data suggest that the HMCp6 peptide fragment might be a novel fragment derived from hemocyanin with a pathogen-resistance function. These results will assist in investigations of the mechanism of these hemocyanin fragments and help to establish strategies for controlling diseases in shrimp.