鱼类抗菌肽的研究进展

抗菌肽是一类广泛存在于生物界的小分子短肽,具有广谱的抗菌、抗病毒、抗寄生虫及抗癌等生物活性。它是鱼类自身先天免疫系统的重要组成部分,结构复杂,种类多样。本文综述了目前发现的鱼类抗菌肽的结构特点、种类、生物学功能、抗菌机制以及基因工程上的研究进展。     

水产动物抗菌肽研究进展

抗菌肽是一类广泛存在于自然界生物体内的小分子多肽物质,为机体固有免疫系统的重要组成部分,对革兰氏阴性菌、革兰氏阳性菌、真菌、病毒和寄生虫等均具有较好的抑制或杀伤作用。抗菌肽具有无污染、无残留、广谱抗菌及不易产生耐药性等特点,有望代替抗生素用于水产动物病原性疾病的防控。近年来,抗菌肽已在水生甲壳动物、水产软体动物、鱼类和两栖类等水产动物中得到报道,但其分类和作用机制有待深入研究。本文对不同水产动物中的抗菌肽进行了分类,对各类抗菌肽的结构特征和功能进行了分析,并从直接杀伤作用、非膜靶向作用和免疫调节作用等三个方面对抗菌肽的免疫作用机制进行了分析,期望能够为今后在水产动物中开展抗菌肽的相关研究和应用提供一定的理论支持。     

Activity of the antimicrobial polypeptide piscidin 2 against fish ectoparasites

Abstract The antiparasitic effects of piscidin 2, an antimicrobial polypeptide (AMPP) first isolated from mast cells of hybrid striped bass, were tested against three protistan ectoparasites of marine fish (the ciliates Cryptocaryon irritans and Trichodina sp., and the dinoflagellate Amyloodinium ocellatum) and one ciliate ectoparasite of freshwater fish (Ichthyophthirius multifiliis). I. multifiliis was the most susceptible parasite, with all theronts killed at 6.3 microg mL(-1) piscidin 2. The most resistant parasite was Trichodina, where a few cells were killed at 12.5 microg mL(-1), but several were still alive even at 100 microg mL(-1). C. irritans was of intermediate sensitivity, with some theronts killed at 12.5 microg mL(-1) and all killed at 25 microg mL(-1). High parasite density apparently exhausted the piscidin 2 before it could attain its maximal effect, but surviving parasites were often visibly damaged. The lower efficacy of piscidin 2 against marine parasites compared with the freshwater ciliate might be related to the inhibitory effects of high sea water cation levels. The tissue concentration of piscidins estimated in healthy hybrid striped bass gill (40 microg mL(-1)) suggests that piscidin 2 is lethal to the parasites tested at physiological concentrations and is thus an important component of innate defence in fish expressing this type of AMPP.     

Effect of biofilm formation on antimicrobial tolerance of Flavobacterium psychrophilum

Treatment of bacterial fish diseases can be complicated by resistant bacterial biofilms harbouring pathogenic bacteria and causing recurrent exposure of fish to infections. In this study, the effect of biofilm formation on antimicrobial tolerance was examined using three bacterial isolates of the fish pathogen Flavobacterium psychrophilum and two antimicrobial agents, oxytetracycline and flumequine, commonly used in aquaculture. Planktonic and biofilm cells were exposed to a minimum inhibitory concentration (MIC), to a 3 × MIC concentration and to an environmental concentration level of each antimicrobial in 96-well microtitre plates after which growth on agar plates was measured. The type strain NCIMB1947 of F. psychrophilum was further used to study the development of antimicrobial resistance in biofilm cells. The results suggest that at high bacterial densities (>10(7) CFU mL(-1)), biofilm cells of F. psychrophilum are less susceptible to antimicrobial agents. Furthermore, the results imply that biofilm cells of F. psychrophilum may rapidly develop resistance to both oxytetracycline and flumequine if exposed to subinhibitory concentrations of these antimicrobials.     

Exogenous polyunsaturated fatty acids (PUFAs) influence permeability,antimicrobial peptide resistance,biofilm formation and membrane phospholipid structure in an A-layer and non-A-layer strain of Aeromonas salmonicida

Aeromonas salmonicida is a Gram-negative bacterium that can infect a wide host range of fish populations, including salmonids and non-salmonids as well as freshwater and marine life. Some strains of A. salmonicida cause the disease furunculosis, which can cause lethargy, intestinal inflammation, ulcers, haemorrhaging and death. The infection is spread through fish-to-fish contact, and the presence of infection can have devastating effects on cultivated fish populations. The purpose of this study was to explore the ability of non-A-layer and A-layer A. salmonicida strains to incorporate polyunsaturated fatty acids (PUFAs) into their lipid profile and test the phenotypic effects thereof. Lipids were extracted from PUFA-exposed cultures and analysed for lipid modification by thin-layer chromatography and ultraperformance liquid chromatography-mass spectrometry, showing A. salmonicida, regardless of A-layer, capable of incorporating all seven of the PUFAs studied. Phenotypic effects were determined through the use of assays that tested for biofilm formation, membrane permeability and cyclic peptide susceptibility. Temperature-dependent effects on biofilm formation were observed, and PUFA exposure showed significant (p < .001) increases in membrane permeability as tested by the uptake of the hydrophobic compounds crystal violet and ethidium bromide. Additionally, some PUFAs elicited modest protection and vulnerability against the membrane-targeting cyclic peptides polymyxin B (PMB) and colistin. The diverse, strain-specific responses to exogenous PUFAs may allude to evolved adaptive strategies that enhance survival, persistence and virulence of non-pathogenic and pathogenic members of bacteria that oscillate between environmental and fish host niches.     

Evidence for synergism of the antimicrobial peptide piscidin 2 with antiparasitic and antioomycete drugs

Piscidins are potent, broad‐spectrum, host‐produced antimicrobial peptides (AMPs) that appear to constitute the most common AMP family in teleost fish. Here, we show that piscidin 2 has potent activity against the water mould Saprolegnia, one of the most important pathogens of freshwater fish. The minimum oomyceticidal concentration (MOC₁₀₀) of piscidin 2 against zoospores of three pathogenic isolates of Saprolegnia ranged from 12.5 to 25.0 μg mL^?1. This piscidin concentration is well within levels that have been estimated to be present in at least some fish (1–32.5 μg mL^?1). In the presence of either copper or malachite green, two drugs commonly used to treat water moulds, there was evidence for partial synergism (PSYN) with piscidin 2. There was also evidence for PSYN after exposure of the ciliate parasite Tetrahymena pyriformis to piscidin 2 plus copper. Our data provide further evidence that piscidins may be an important host defence against skin and gill pathogens and that the piscidin levels in host tissue might influence the success of drug treatments.     

重组抗菌肽的制备及其对水产养殖中常见病原菌的抑菌效果

采用YEPD培养基诱导发酵生产重组抗菌肽。经 72h培养 ,由重组酵母表达并分泌到培养基中的抗菌肽活性可高达 5 0 0 0U/ml。用纸片扩散法和试管稀释法测定重组抗菌肽对水产养殖常见病原菌的抑菌效果。结果表明 ,抗菌肽对水产养殖常见病原菌嗜水气单胞菌 (Aeromonashydrophila)、梅氏弧菌 (Vibriometschnikovi)、迟钝爱德华氏菌 (Edwardsiellatarda)及温和气单胞菌 (A .sobria)有较好的抑菌效果 ,最小抑菌质量浓度为 4 μg/ml、杀菌质量浓度为 8μg/ml。对抗菌肽抑菌过程的超微结构观察表明 ,抗菌肽的抑菌方式是通过作用于细菌的细胞膜而导致菌体裂解死亡。本研究目的在于探讨基因工程抗菌肽在水产养殖中的应用。     

Effects of antimicrobial peptides on growth,feed utilization,serum biochemical indices and disease resistance of juvenile shrimp,Litopenaeus vannamei

This experiment was conducted to assess the effects of antimicrobial peptides at different levels (Diet 1 (0%), Diet 2 (0.1%), Diet 3 (0.2%), Diet 4 (0.4%), Diet 5 (0.6%) and Diet 6 (1%)), on growth, serum biochemical indices and antioxidant effect, feed utilization and disease resistance in Litopeneaus vannamei. There were four replicates in each group in the experiment (mean weight = 0.21 ± 0.00 g) and also fed with their respective diets for 8 weeks. Growth performance compared with the control group (0%) significantly increased at first and then decreased among treatment groups (p < .05) whereas the survival rate ranged from 78% to 96%. No significant differences were observed in terms of moisture, crude protein and ash content, but there was a significant increase in crude lipid (p < .05). In serum, acid phosphatase, alkaline phosphatase, polyphenol oxidase, triglyceride, glucose and total cholesterol changed as compared with 0%. Total antioxidant capacity, glutathione peroxidase, catalase, superoxide dismutase and malondialdehyde were also different from 0%. Disease resistance was increased in shrimp among treatment groups with 0.4% recording the lowest mortality percentage of 37% after the challenge test. The results from the present study suggested that supplementation of AMP at 0.4% in shrimp diet can improve growth performance, antioxidants activities and innate immune response of Pacific whiteleg shrimp.     

Comparative activity and stability under salinity conditions of different antimicrobial peptides isolated from aquatic animals

This study reports the in vitro activity of six antimicrobial peptides (AMPs) produced by aquatic animals (most marine invertebrates): tachyplesin (Tach), magainin (Mag), clavanin (Clav), penaeidin (Pen), mytilin (Myt) and antilipopolysaccharide factor (ALF) against marine vibrios, filamentous fungi and yeast. Their stability under salinity conditions and seawater was also examined. The results showed that Mag, Myt and especially Tach and ALF (minimum inhibitory concentration<1.5 μM) had a potent activity against all tested vibrio species, whereas Clav and Pen were ineffective (up to 50 μM). With respect to the antifungal activity, each AMP had a different potency according to the fungal species. In general terms, Tach was the most potent peptide, followed by Mag. Interestingly, Tach, Myt and ALF had a significant effect on the filamentous fungus Fusarium solani that could be pathogenic to marine organisms. All AMPs had a tendency to decrease or lose their activity at high salinity (>225 mM NaCl). Tach and Myt were the most stable peptides, maintaining significant activity under seawater salinity (450 mM). Curiously, all peptides lost their effect under seawater conditions. The results suggest that Tach, ALF and Myt are the most promising candidates for potential therapeutic use in farmed-marine species, because all have a significant and broad antimicrobial activity maintained at high salinity.     

棕点石斑鱼Piscidin样肽基因的克隆及其成熟肽的原核表达

以简并引物通过RT-PCR从重要经济海水鱼类棕点石斑鱼Epinephelus fuscoguttatus白细胞中扩增出一条388bp具有完整编码框的cDNA序列。Blast分析初步显示,该序列属于鱼类抗菌肽Piscidin家族的一员,将其命名为棕点石斑鱼Piscidin样肽(GU592793),推导氨基酸序列具有界定鱼类Piscidin的以下共同特征:(1)预测的信号肽序列与其他Piscidin的同源性较高,在60%~95%之间;(2)推测的成熟肽N端6个氨基酸残基富含Phe、Ile及His;(3)成熟肽第8个和第13个氨基酸位点均为Gly。NJ进化树分析表明,该cDNA推导的氨基酸序列与赤点石斑鱼Epinephelusakaarapiscidin-like peptide(ACE78290)、斜带石斑鱼Epinephelus coioidesPiscidin-like peptide(ACE78291)以高达81%的支持率聚为一支。也表明本研究成功克隆到棕点石斑鱼首条PiscidincDNA序列。推测的成熟肽为两亲性阳离子肽,等电点12.48,Schiffer-Edmunson预测其二级结构呈α螺旋构型。将...     

饲料中添加抗菌肽和枯草芽孢杆菌对花鲈生长和非特异性免疫功能的影响

在基础饲料中分别添加300 mg/kg的抗菌肽和200 mg/kg的枯草芽孢杆菌(Bacillus subtilis),连续投喂花鲈(Lateolabrax japonicus)60 d,测定其特定生长率、存活率,并分别于试验的20 d、40 d和60 d测定肝胰脏、头肾、鳃的过氧化氢酶(CAT)、超氧化物歧化酶(SOD)以及血清溶菌酶(LSZ)和一氧化氮合酶(NOS)活力水平。结果显示:试验组花鲈的特定生长率和存活率与对照组间均无显著差异(P>0.05)。抗菌肽组所测组织CAT、SOD以及血清LSZ、NOS活性水平在20 d时均显著高于对照组(P<0.05),40 d时以上指标(头肾SOD除外)与对照组间差异不显著(P>0.05)。枯草芽孢杆菌组所测组织CAT、SOD以及血清LSZ活性水平在20 d时均显著高于对照组(P<0.05),60 d时以上指标(头肾SOD除外)与对照组间差异不显著(P>0.05),而整个过程血清NOS活性水平与对照组不存在显著性差异(P>0.05)。结果表明:两种添加剂可增强机体消除活性氧自由基的能力,提高机体血清溶菌酶活性水平。此外,抗菌肽对花鲈血清一氧化氮合酶系统具有诱导调节作用。     

中国对虾中抗菌短肽的分离纯化与功能分析

以水产病害的病原菌为目标菌，从中国对虾（Penaeuschinesis）体液中分离筛选抗菌肽，通过Sephadex G-50、RP-HPLC等分离纯化技术，得到一种抑制水产病原菌的抗菌肽PC-V-2。经初步鉴定，该抗菌肽具有广谱抗性，不仅抗鲁克氏耶尔森氏菌（Yersinia ruckeri），而且对金黄色葡萄球菌（Staphyloccus auleus）、枯草杆菌（Bacillus subttilis）等革兰氏阳性菌，大肠杆菌（Escherichia coli）等革兰氏阴性菌和白色念珠菌（Candida albicans）等真菌也有较强的抗性。另外，该抗菌肽还有一定的抑制凝血的活性，但没有检测到溶血和丝氨酸蛋白酶抑制活性。经MALDI-TOF质谱分析，其分子量为1848D，但其序列及其他生物特性还有待进一步研究。     

鱼粉水解物中小肽对幼龄草鱼生长性能的影响

用150尾3.74±0.23 g的草鱼作日粮中鱼粉水解物中小肽对幼龄草鱼生长性能影响研究。实验分为2 组,每组75尾鱼,每25尾鱼饲于0.25m3的水族箱中,每组设3个平行水族箱。试验组日粮中添加0.5%鱼粉水解物 0.5%鱼粉,对照组为1%鱼粉,试验期56 d。结果表明,试验组草鱼其特定生长率、蛋白保留效率、饲料系数和血浆中镁含量与小肽总量均显著高于对照组(P<0.05)。草鱼日粮中添加一定比例的鱼粉水解物小肽可提高饲料表观消化率和蛋白消化率,增加血液循环中生物活性肽的含量,增加体内氮沉积,减少肝胰脏和肠系膜脂肪储积,从而提高机体对日粮中蛋白质的利用率。     

Effects of antimicrobial peptides on growth,morphology of foregut villi and related genes mRNA expression in the common carp (Cyprinus carpio)

A 60‐day feeding trial was conducted to examine the effects of different levels (0, 100, 200, 400 and 600 mg/kg) of antimicrobial peptides on growth, protease activity of foregut, the morphology of foregut villi and related genes mRNA expression level in the common carp (Cyprinus carpio). The results showed that the feed of antimicrobial peptides promote common carp growth, and the optimal dosage of antimicrobial peptides is 200–333 mg/kg in the common carp feed. The protease activity of 200 and 400 mg/kg groups were significantly higher than the control and other groups (p < 0.05). The foregut villus height with 100, 200 and 400 mg/kg antimicrobial peptide groups were significantly higher than control group (p < 0.05). The crypt depth of 200 and 400 mg/kg antimicrobial peptide groups were significantly lower than control group (p < 0.05). The ratio of villus height and crypt depth of 100, 200 and 400 mg/kg antimicrobial peptide groups were significantly higher than control group (p < 0.05). The ratio with 600 mg/kg group was significantly lower than the control group (p < 0.05). The IGF‐I gene expression level of 200 mg/kg and 400 mg/kg groups were significantly higher than the control group and 600 mg/kg group (p < 0.05). The IL‐1β gene expression level of 100 mg/kg and 200 mg/kg groups were significantly higher than the control group (p < 0.05). These results indicated up‐regulation of growth and immune related genes in antimicrobial peptides fed common carp. Correlation analysis showed that IGF‐I mRNA and IL‐1β mRNA were positively correlated with SGR. IL‐1β mRNA and FCR were significantly negative correlated. It indicated that growth and immune gene common regulated the growth of the carp under antimicrobial peptides intervention. In conclusion, antimicrobial peptides can improve growth and related genes mRNA expression in the common carp. Further studies using molecular biological technique or immunologic methods are required to conclude that antimicrobial peptides are beneficial in common carp.     

饲料中添加重组抗菌肽对吉富罗非鱼生长性能及免疫力的影响

以吉富罗非鱼(genetically improved farmed tilapia,GIFT)幼鱼(体质量10.68 g±0.53 g)为研究对象,在实验饲料中分别添加0、5、10、20、50 mg/kg重组中国明对虾抗菌肽以及100 mg/kg氟苯尼考,并于实验结束时进行注射攻毒(嗜水气单胞菌).通过8周的养殖实验...     

生物活性肽对断奶仔猪生长性能影响的饲养对比试验

选择平均体重约9.5kg左右的三元杂交生长猪60头,随机分成3个处理组,每个处理设2个重复,每个重复10头仔猪。试验1组和试验2组在日粮中添加0.3%的小肽制剂,经过29d试验,与对照组相比平均日增重分别提高11.9%(P<0.05)和12.9%(P<0.05),料肉比降低10.6%(P<0.05)和11.06%(P<0.05),生长猪腹泻率分别降低25%(P<0.05)和20%(P<0.05)。     

凡纳滨对虾细菌性红体病病原的分子特征与耐药性

为探明引起凡纳滨对虾细菌性红体病的病原,从病虾肝胰脏分离得到10株优势菌,经回归感染实验证实其为引起此次红体病的病原菌.Vitek与16S rRNA序列分析显示,分离株均为副溶血弧菌.基于dnaE-gyrB-recA-dtdS-pntA-pyrC-tnaA的多位点序列分型(multilocus sequence typing,MLST)表明,这些菌株形成3个新序列型(ST),其中1株为ST413,7株为ST414,2株为ST415;ST413包含新等位基因型recA-166与tnaA-121,ST414则含有新等位基因型gyrB-219.MLST结果提示,这些副溶血弧菌分离株并非来自单一克隆,呈现出一定水平的分子多样性.但这些菌株均含有大流行群(PG)的分子标记toxRS与VPA1168,并具有相同的毒力基因构成(tlh+ tdhtrh-T3SS1+T3SS2-)与耐药谱,其tdh与trh的缺失并未影响细菌对凡纳滨对虾的致病力.综上所述,引起此次凡纳滨对虾红体病的10株副溶血弧菌可能为PG的不同变异株.     

人工鱼礁表面分离细菌形成单一生物被膜对厚壳贻贝稚贝附着的影响

为探讨存在于人工鱼礁表面的海洋细菌与贝类附着之间的互作关系,本研究从自然海域中的人工鱼礁表面上分离了9株海洋细菌,并分别构建单一细菌生物被膜,探索不同细菌种属形成的生物被膜特性与厚壳贻贝(Mytilus coruscus)稚贝附着之间的关系。结果显示,9株人工鱼礁表面细菌形成的生物被膜对厚壳贻贝稚贝附着的诱导活性存在显著差异,其中,Mesoflavibacter sp.2对稚贝附着的诱导活性最高,Phaeobacter sp.2的诱导活性最低。Sutcliffiella sp.1和Jeotgalibacillus sp.1的细菌密度与诱导活性呈显著正相关,Cytobacillus sp.1和Phaeobacter sp.2的细菌密度与诱导活性呈显著负相关。通过比较分析Mesoflavibacter sp.2和Phaeobacter sp.2生物被膜的蛋白质及多糖含量发现,生物被膜对厚壳贻贝稚贝附着的诱导活性与多糖含量呈显著负相关,与蛋白质含量呈正相关。本研究初步探索了人工鱼礁表面细菌形成的生物被膜对厚壳贻贝稚贝附着的影响,为后续在自然海区进一步解析人工鱼礁表面生物被膜与海洋无脊椎动物附着的互作关系具有重要理论研究意义,同时,对于人工鱼礁表面海洋生物附着机制的研究具有重要的实践价值。