黄斑蓝子鱼（Siganusoramin）皮肤黏液对多种病原菌和寄生虫的抑制杀灭作用
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摘要
鱼类体表黏液是鱼类先天性免疫重要组分,是抵御外界病害的第一道屏障。黏液中除了黏液糖蛋白以外,还有许多具有免疫活性物质,其中抗菌蛋白/肽是其中重要的一类。抗菌蛋白/肽广泛存在于动物、植物和微生物中,它们具有不同程度的抗细菌、真菌和寄生虫的活性。
本论文研究了黄斑蓝子鱼皮肤黏液对细菌、真菌、寄生虫的抗性作用。通过和其他鱼类比较发现黄斑蓝子鱼的黏液除了对大肠杆菌没有抑制作用外,对其他8种供试细菌都有明显的生长抑制作用。黄斑蓝子鱼黏液对金黄色葡萄球菌、霍乱弧菌、海豚链球菌、温和气单胞菌、溶藻弧菌和副溶血弧菌的最低抑菌浓度分别为0.16mg/ml、0.16mg/ml、0.08 mg/ml、0.63 mg/ml、0.63 mg/ml和0.63 mg/ml。对小麦赤霉菌和黑曲霉菌无明显抑制杀灭效果。
在抗寄生虫的体外作用实验中,黄斑蓝子鱼黏液具有明显的杀虫效果。对于刺激隐核虫来说,60min后黄斑蓝子鱼黏液使幼虫全部死亡的最低蛋白浓度为4mg/ml,3mg/ml浓度的黏液幼虫死亡率会随着时间的推移有所上升,最终达到43.33±5.84%,1mg/ml和2mg/ml的作用效果不明显。死亡幼虫的形态发生很大的变化,会出现脱纤毛、虫体肿胀、外膜破裂和内容物泄漏等现象,滋养体在作用后也会出现破裂和内容物泄漏的现象。对多子小瓜虫来说,60min后使幼虫全部死亡的最低蛋白浓度为5mg/ml,3mg/ml和4mg/ml使幼虫的死亡率达到64.00±2.92%和45.67±5.84%,1mg/ml和2mg/ml的作用效果不明显,其死亡幼虫形态会出现脱纤毛、虫体肿胀、外膜破裂和内容物泄漏等现象。对布氏锥虫来说,60min后使所有的锥虫死亡的最低蛋白浓度为3mg/ml,0.5mg/ml的作用效果不明显。
黄斑蓝子鱼黏液在60℃作用10min后活性有所增强,80℃作用10min后完全失活。黏液在酸性条件下活性增强,在pH 3.0、4.0处的活性最强,碱性条件下活性减弱。胰蛋白酶使黏液的抗菌活性降低,溶菌酶和蛋白酶K不会降低其活性。
通过凝胶过滤层析和反相高效液相层析对黄斑蓝子鱼黏液抗菌抗寄生虫活性物质进行初步分离,发现在反相高效液相层析的洗脱峰中有两个峰有抗菌活性,有一个峰有抗寄生虫活性。
The mucus of fish skin epithelium is a vital component in fish innate immunolgy, which is the first barrier against diseases in the entironment. Besides glycoproteins, there is plenty of immunologic active material, one of which is antimicrobal proteins/peptides(AMPs). AMPs are exsisted widespread in annimals, plants and microorganism, which have antibacterial,antifungal and antiparasite activities to different degree.
Our research was about the antibacterial, antifugal and antiparasite activities of skin mucus of rabbitfish(S.oramin), compared with other 4 species of fish (E.coioides, S.ocellatus, P.cinctus and T.ovatus). S.aureus, A.sobria, V.cholerae, V.parahaemolyticus, V.alginolyticu, S.iniae,Nocardia and Pdsubsp.piscicida, were inhibited by the mucus of rabbitfish,except E.coli. The minimal inhibitory concentrations of mucus of rabbitfish were 0.16mg/ml, 0.16mg/ml, 0.08 mg/ml, 0.63 mg/ml, 0.63 mg/ml and 0.63 mg/ml to S.aureus, V.cholerae, S.iniae, A.sobria, V.alginolyticu and V.parahaemolyticus. All of 5 species mucus have no antifungal activity against G.saubinetii and A.niger.
The mucus of rabbitfish had marked antiparasites effect in vitro. To C.irritans, all of theronts were killed at 4mg/ml rabbitfish mucus in 60min. The mortality was increasing as time went on and to 43.33±5.84% finally at 3mg/ml.The antiparasite activity was not obvious at 1mg/ml and 2mg/ml.Under the light microscopy, we could see the shape of C.irritans theronts changing massively, such as cilia falling off, rapture and content leakage, and the mucus also made trophonts rapture and leakage. To I.multifilis, all of theronts were killed at 5mg/ml rabbitfish mucus in 60min. The mortalities at 3mg/ml and 4mg/ml were 64.00±2.92% and 45.67±5.84% respectively. The antiparasite activity was not obvious at 1mg/ml and 2mg/ml. The phenomenon under the light microscopy was similar to C.irritans. To T.brucei all of cells were killed at 3mg/ml rabbitfish mucus in 60min, The antiparasite activity was not obvious at 0.5mg/ml.
The activity of rabbitfish mucus was enhanced by heating at 60℃for 10min, and the mucus was inactive completely by heating at 80℃for 10min. The activity of mucus was enhanced in acid solution, especially at pH3.0、4.0, and was weakened in alkaline solution. Trypsin could weakened the activity of mucus, but lysozyme and protease K could not.
Antibacterial and antiparasites material was purificated preliminary via gel filtration chromatography and RP-HPLC. Two elution peaks in RP-HPLC had antibacterial activity, one of which the had antiparasite activity.
本论文研究了黄斑蓝子鱼皮肤黏液对细菌、真菌、寄生虫的抗性作用。通过和其他鱼类比较发现黄斑蓝子鱼的黏液除了对大肠杆菌没有抑制作用外,对其他8种供试细菌都有明显的生长抑制作用。黄斑蓝子鱼黏液对金黄色葡萄球菌、霍乱弧菌、海豚链球菌、温和气单胞菌、溶藻弧菌和副溶血弧菌的最低抑菌浓度分别为0.16mg/ml、0.16mg/ml、0.08 mg/ml、0.63 mg/ml、0.63 mg/ml和0.63 mg/ml。对小麦赤霉菌和黑曲霉菌无明显抑制杀灭效果。
在抗寄生虫的体外作用实验中,黄斑蓝子鱼黏液具有明显的杀虫效果。对于刺激隐核虫来说,60min后黄斑蓝子鱼黏液使幼虫全部死亡的最低蛋白浓度为4mg/ml,3mg/ml浓度的黏液幼虫死亡率会随着时间的推移有所上升,最终达到43.33±5.84%,1mg/ml和2mg/ml的作用效果不明显。死亡幼虫的形态发生很大的变化,会出现脱纤毛、虫体肿胀、外膜破裂和内容物泄漏等现象,滋养体在作用后也会出现破裂和内容物泄漏的现象。对多子小瓜虫来说,60min后使幼虫全部死亡的最低蛋白浓度为5mg/ml,3mg/ml和4mg/ml使幼虫的死亡率达到64.00±2.92%和45.67±5.84%,1mg/ml和2mg/ml的作用效果不明显,其死亡幼虫形态会出现脱纤毛、虫体肿胀、外膜破裂和内容物泄漏等现象。对布氏锥虫来说,60min后使所有的锥虫死亡的最低蛋白浓度为3mg/ml,0.5mg/ml的作用效果不明显。
黄斑蓝子鱼黏液在60℃作用10min后活性有所增强,80℃作用10min后完全失活。黏液在酸性条件下活性增强,在pH 3.0、4.0处的活性最强,碱性条件下活性减弱。胰蛋白酶使黏液的抗菌活性降低,溶菌酶和蛋白酶K不会降低其活性。
通过凝胶过滤层析和反相高效液相层析对黄斑蓝子鱼黏液抗菌抗寄生虫活性物质进行初步分离,发现在反相高效液相层析的洗脱峰中有两个峰有抗菌活性,有一个峰有抗寄生虫活性。
The mucus of fish skin epithelium is a vital component in fish innate immunolgy, which is the first barrier against diseases in the entironment. Besides glycoproteins, there is plenty of immunologic active material, one of which is antimicrobal proteins/peptides(AMPs). AMPs are exsisted widespread in annimals, plants and microorganism, which have antibacterial,antifungal and antiparasite activities to different degree.
Our research was about the antibacterial, antifugal and antiparasite activities of skin mucus of rabbitfish(S.oramin), compared with other 4 species of fish (E.coioides, S.ocellatus, P.cinctus and T.ovatus). S.aureus, A.sobria, V.cholerae, V.parahaemolyticus, V.alginolyticu, S.iniae,Nocardia and Pdsubsp.piscicida, were inhibited by the mucus of rabbitfish,except E.coli. The minimal inhibitory concentrations of mucus of rabbitfish were 0.16mg/ml, 0.16mg/ml, 0.08 mg/ml, 0.63 mg/ml, 0.63 mg/ml and 0.63 mg/ml to S.aureus, V.cholerae, S.iniae, A.sobria, V.alginolyticu and V.parahaemolyticus. All of 5 species mucus have no antifungal activity against G.saubinetii and A.niger.
The mucus of rabbitfish had marked antiparasites effect in vitro. To C.irritans, all of theronts were killed at 4mg/ml rabbitfish mucus in 60min. The mortality was increasing as time went on and to 43.33±5.84% finally at 3mg/ml.The antiparasite activity was not obvious at 1mg/ml and 2mg/ml.Under the light microscopy, we could see the shape of C.irritans theronts changing massively, such as cilia falling off, rapture and content leakage, and the mucus also made trophonts rapture and leakage. To I.multifilis, all of theronts were killed at 5mg/ml rabbitfish mucus in 60min. The mortalities at 3mg/ml and 4mg/ml were 64.00±2.92% and 45.67±5.84% respectively. The antiparasite activity was not obvious at 1mg/ml and 2mg/ml. The phenomenon under the light microscopy was similar to C.irritans. To T.brucei all of cells were killed at 3mg/ml rabbitfish mucus in 60min, The antiparasite activity was not obvious at 0.5mg/ml.
The activity of rabbitfish mucus was enhanced by heating at 60℃for 10min, and the mucus was inactive completely by heating at 80℃for 10min. The activity of mucus was enhanced in acid solution, especially at pH3.0、4.0, and was weakened in alkaline solution. Trypsin could weakened the activity of mucus, but lysozyme and protease K could not.
Antibacterial and antiparasites material was purificated preliminary via gel filtration chromatography and RP-HPLC. Two elution peaks in RP-HPLC had antibacterial activity, one of which the had antiparasite activity.
引文
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何介华,邹思湘,陆承平.团头鲂血清抗病毒半乳糖凝集素样蛋白的分离与鉴定.水产学报, 2003, 27:474-479
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李哗,苏秀榕,李太武.鲍血淋巴抗菌肽的研究.食品科学, 2005, 26:67-70
梁英,黄文树,关瑞章等.鱼类表皮黏液抗菌蛋白/肽研究进展.水生生物学, 2009, 33:963-969
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马强,刘静.蓝子鱼科的系统研究概况及我国蓝子鱼科的研究展望.南方水产, 2006, 2:68-74
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