耳鲍血淋巴抗菌活性的研究

用溶藻弧菌诱导后,收集耳鲍血淋巴,利用微量测定法进行抗菌活性分析.结果发现,耳鲍血淋巴具有抗革兰氏阴性菌-溶藻弧菌、副溶血弧菌、河流弧菌、嗜水气单胞菌、大肠杆菌的活性,对革兰氏阳性菌的抑制活性较低.利用胰蛋白酶对耳鲍血淋巴水解后,其抗菌活性消失,表明其中抗菌成分为蛋白质.将耳鲍血淋巴在不同温度(37～95 ℃)和不同pH(2.5～5.0)下保温,发现其抗菌活性成分对热及酸耐受性较强.耳鲍血淋巴在低浓度无溶血活性.该抗菌组分在耳鲍的免疫防御中具有一定的作用.     

马氏珠母贝人工育苗病害防治试验

马氏珠母贝（Pinctada martensii）育苗技术已经发展得比较成熟,近几年来,作者调查发现马氏贝种苗人工培育的过程,最常见且影响最大的是马氏珠母贝D形幼虫阶段的粘连病,其发病频度约占马氏珠母贝种苗人工培育阶段病害发生的60％.马氏珠母贝D形幼虫一经粘连就会下沉以致死亡,造成育苗失败.有研究发现粘连病是一种弧菌病,病原：鳗弧菌（V.anguillarum）和溶藻酸弧菌（V.alginolyticus）等.此病感染很快,幼虫感染病菌4～5h后就出现症状,8h后开始死亡,幼虫组织坏死和消散,一般在18h内幼虫100％死亡,幼虫往往发生面盘解体以后立即下沉死亡.这是贝类育苗中危害最大的一种疾病,是育苗成败的关键.     

珍珠贝外套膜酶解产物促进皮肤创伤愈合效果研究

为进一步拓宽马氏珠母贝(Pinctada martensii)的高值化利用途径,文章探讨了马氏珠母贝外套膜酶解产物体外抗菌活性、促凝血作用以及对小鼠软组织开放性创伤愈合效果。结果表明,珍珠贝外套膜酶解产物具有一定的促凝血作用,但对13种测试菌无抑制作用;涂抹给药6 d后,给药组小鼠的创伤愈合率显著高于阴性对照组(P0.05),与阳性对照组无显著差异(P0.05);给药组的第14天愈合率达100%,疤痕缩小率显著高于阴性对照组(P0.05)。生化指标测试结果显示,与2个对照组相比,马氏珠母贝外套膜酶解产物能够抑制炎症因子IL-6生成;与阴性对照组相比,其可极显著促进IL-10分泌;与阴性对照组相比,给药组皮肤组织中FGF-2含量无显著性差异(P0.05),阳性对照组EGF含量显著高于其他组(P0.05);而TGF-β和CCND1含量均显著高于阴性对照组(P0.05)。综上,马氏珠母贝外套膜酶解产物具有一定促凝血、抑制炎症作用并促进生长因子分泌,从而加快小鼠软组织开放性创伤愈合,对浅表瘢痕增生具有一定的抑制作用。     

马氏珠母贝正常贝肉与黑壳病贝肉成分对比

为了研究黑壳病造成马氏珠母贝肉成份的变化,首先采用水蒸气蒸馏法分别提取正常马氏珠母贝和黑壳病马氏珠母贝肉的挥发性成份进行分析,正常马氏珠母贝肉检测出58种,黑壳病贝肉检测出59种。但黑壳病贝肉中仅有7种化合物与正常贝肉的挥发性成分相同。在黑壳病贝中,含量较高的成分有二十一烷和十八醛,分别为18.52%和17.95%,但在正常贝肉未检出。其次,采用茚三酮作柱后衍生试剂测定氨基酸的方法检测正常马氏珠母贝和患黑壳病的马氏珠母贝肉中牛磺酸的含量,结果分别为72.47mg/g、72.75mg/g,牛磺酸含量基本一致。通过分析黑壳病引起马氏珠母贝体内挥发性成分和牛黄酸成分变化,为研究马氏珠母贝患黑壳病的病变机理提供一定的科学依据。     

术前限食处理对马氏珠母贝鳃和肝脏抗氧化功能的影响

为更好地了解术前处理对马氏珠母贝生理状态的影响,进而为改进育珠贝的术前处理方法、培育优质珍珠提供参考,在实验室养殖条件下,研究了术前限食处理对马氏珠母贝鳃和肝脏超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽S转移酶(GST)活性的影响。试验设1 d投喂1次、2 d投喂1次两个限食处理组,对照组1 d投喂2次。在试验开始和第1、2、3、4周取样检测鳃和肝脏相关抗氧化酶的活性。结果显示:限食1周后,马氏珠母贝鳃和肝脏SOD、CAT和GST的活性均随限食强度的增大呈下降趋势;2个限食处理组的抗氧化酶活性较对照组有明显下降,且差异显著(P0.05);2 d投喂1次组各项酶的水平均显著低于对照组,这种趋势一直延续至第4周。据此推断,术前限食可以显著降低插核手术贝的生理机能和免疫活性。     

马氏珠母贝TLR6基因的克隆、序列分析与表达

为了探究TLR6(Toll like receptor 6)在马氏珠母贝免疫反应中的作用,实验采用c DNA末端快速扩增(RACE)技术获得了马氏珠母贝TLR6基因(Pm-TLR6)c DNA全长序列,并且运用实时荧光定量PCR(q RT-PCR)技术检测了Pm-TLR6在马氏珠母贝各组织中的表达情况、哈维氏弧菌刺激后和植核移植后血淋巴中的表达模式。结果显示:Pm-TLR6c DNA全长2295 bp,其中5′非编码区(UTR)长94 bp,3′UTR长89 bp,开放阅读框(ORF)长2112 bp,编码703个氨基酸;多序列比对结果表明物种间TLR6具有较高的保守性;PmTLR6具有跨膜域、富含亮氨酸的重复序列(LRRs)和TIR域,符合TLRs家族特征。q RTPCR数据分析表明,Pm-TLR6在马氏珠母贝肝胰脏、血细胞、鳃、性腺、闭壳肌、外套膜中均有表达,其中肝胰脏中表达量最高;哈维氏弧菌刺激后,Pm-TLR6在2 h表达上调,约为对照组(0 h)的9倍,随后的6 h恢复至正常水平,直至16 h表达水平开始回升并于24 h达到最大表达量,是对照组的29.4倍,具有显著性差异;植核移植实验结果显示PmTLR6在植核后5和10 d表达水平没有显著性变化,15和20 d表达量出现上升趋势,但差异不显著,最后于30 d达到最大值,约为空白对照组(0 d)的5倍,具有显著性差异。研究表明,Pm-TLR6可能在马氏珠母贝免疫防御反应中担任着重要的角色。     

马氏珠母贝Rab7基因的克隆及其表达特征分析

Rab家族基因在囊泡的形成、转运、黏附、锚定和融合等各个阶段发挥重要作用。在已有的马氏珠母贝RabcDNA片段的基础上,采用RACE方法克隆了该基因的全长。cDNA长度2519bp,编码区长618bp,编码206个氨基酸。编码的蛋白质序列分析表明,该基因具有Rab家族的全部保守结构,与人类等其他生物的Rab7亚家族的同源性最高。RT-PCR结果显示,该基因在马氏珠母贝的鳃、足和胃组织中没有扩增产物,在卵、感染和未感染凿贝才女虫Polydora ciliata的马氏珠母贝的肝脏和血液中均检测到了与预期大小相符的扩增产物,存在组织差异性表达。     

溶藻弧菌诱导马氏珠母贝血淋巴cDNA差减文库的构建及分析

为了探讨马氏珠母贝免疫防御机制,筛选免疫防御相关功能基因,以致病性溶藻弧菌人工感染的马氏珠母贝血淋巴为材料,采用抑制性差减杂交(SSH)技术,构建了溶藻弧菌诱导的马氏珠母贝血淋巴cDNA差减文库;以马氏珠母贝管家基因β-actin作为差减指标检测该文库的差减效率;对选取的600个阳性克隆进行测序及生物信息学分析。结果显示,该文库的差减效率可达210倍;PCR阳性检测显示差减片段为100~750 bp,对随机挑取600个克隆的测序结果显示,共获得414个有效EST序列;通过BLAST同源性比对,有167个EST序列(占40.34%)与NCBI数据库中已知功能蛋白质具较高同源性,其中7个EST序列(占1.69%)与免疫防御相关基因同源;此外,204个EST(占49.28%)在数据库中未发现同源序列。研究表明,SSH技术能有效富集马氏珠母贝血淋巴差异表达基因,研究结果为探索马氏珠母贝免疫基因作用机理和调控机制奠定了基础。     

星斑川鲽免疫相关组织抗菌活性的研究

鱼类黏液中含有许多非特异性抗菌活性成分,在自然条件下可抵抗微量病原菌的侵害,是鱼类先天性免疫系统的重要组成部分。本研究对新兴养殖鱼类星斑川鲽免疫相关组织脾脏、肾脏、后肠及皮肤黏液中抗菌物质的抗菌活性进行了研究。结果表明,星斑川鲽各免疫相关组织的提取物具有抗鳗弧菌、溶藻弧菌、副溶血弧菌和爱德华氏菌的活性。各组织的抗菌物质对温度不敏感,分别在37、50、70和95℃不同温度中处理10 min,95℃处理的样品抗菌活性显著下降,而其他温度处理后抗菌活性则变化不明显。体表黏液抗菌物质的抗菌活性在酸性条件下强于碱性条件。溶菌酶与黏液及各组织的抗菌物质作用后,其抗菌活性略有下降,胰蛋白酶和蛋白酶K处理后则没有明显的变化,并且黏液及各组织的抗菌物质具有较高的溶血活性。经凝胶过滤层析的初步鉴定结果表明,各组织的抗菌活性物质可能是分子量较小的物质。     

马氏珠母贝混养紫海胆对污损生物防除效果的研究

利用我国南方紫海胆(Anthocidaris crassispina)与大亚湾马氏珠母贝(Pinctada martensi)进行不同比例混养,探讨了紫海胆对马氏珠母贝贝体和养殖网笼污损生物的防除效果,以及紫海胆和马氏珠母贝的生长状况。试验设混养组(PA3、PA6、PA9)和对照组(P),即在养殖马氏珠母贝30只/层的基础上,分别混养紫海胆3、6、9、0个/层;另设紫海胆单养组(A3、A6、A9),养殖密度分别为3、6、9个/层。试验结果显示：各混养组及P组间马氏珠母贝的存活率无显著差异(P>0.05),混养组紫海胆的存活率均接近100%,组间无显著差异(P>0.05);PA6、PA9组马氏珠母贝的特定生长率大于P组(P<0.05),而PA3组与P组之间差异不显著(P>0.05);就马氏珠母贝的壳长日平均增长率而言,PA3、PA9组与P组间无显著差异(P>0.05),但PA6组显著大于P组和PA3组(P<0.05);PA6、PA9组马氏珠母贝的肥满度显著大于P组(P<0.05),PA3组与P组间无显著差异(P>0.05);混养与单养各组之...     

几种海藻多酚对鱼类致病菌的抑菌性研究

用平板生长抑制法对角叉菜、海蒿子及鼠尾藻等藻体中的多酚化合物进行抑菌性试验,观察其对海神弧菌、巴西弧菌、徐氏弧菌、查格斯氏弧菌、鳗弧菌、波氏弧菌及哈维氏弧菌的抑菌效果。试验结果表明,不同来源海藻多酚对受试海洋弧菌的抑制能力具有显著的差异(P0.05);多酚含量越高,抑菌能力越强;多酚含量和多酚来源具有显著的交互效应(P0.05),随着多酚含量的增加,抑制差异显著增强;同一来源多酚对不同受试海洋弧菌的抑制差异性不显著(P0.05)。     

Exploration of the antibacterial proteins in pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> induced by bacterial inoculation

The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 10¹² colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.     

Inhibition of Vibrio spp. by 2‐Hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate of Marine Cyanobacterium Leptolyngbya sp. LT19

Seven marine cyanobacteria were isolated from two regions of the Gulf of Thailand and evaluated by the agar diffusion method for antibacterial activity. Inhibitory compound was purified from the crude methanol extract and its structure was elucidated based on extensive spectroscopic analysis, including IR, 1D and 2D NMR spectra as well as mass spectrometry. A novel antimicrobial compound produced by the marine cyanobacterium Leptolyngbya sp. LT19 was identified to be a 2‐hydroxyethyl‐11‐hydroxyhexadec‐9‐enoate which has so far never been reported in microorganisms. Biological assays revealed that this novel compound exhibited antibacterial activities against the Gram‐negative, persistent shrimp pathogens, Vibrio harveyi and V. parahaemolyticus with minimal inhibitory concentration of 250–1000 and 350–1000 μg mL^?1 respectively.     

皱纹盘鲍肽聚糖识别蛋白在免疫防御中的作用

本研究从皱纹盘鲍(Haliotis discus hannai)中鉴定并克隆了一种肽聚糖识别蛋白(PGRP),命名为HdPGRP。HdPGRP的cDNA全长为1467 bp,共编码354个氨基酸,其中含有1个信号肽(1~18氨基酸)、1个SH3b结构域(93~160氨基酸)、1个PGRP结构域(179~322氨基酸)和1个Ami_2结构域(191~332氨基酸)。此外,在HdPGRP序列中发现了4个保守的Zn2+结合位点(H209、Y255、H318和C330)以及5个保守的酰胺酶催化位点(H209、Y255、H318、T328和C330)。经多序列比对和系统发育树分析,表明HdPGRP属于短型PGRP家族成员。在健康鲍鱼中,hdpgrp主要在肝胰腺中表达,其次依次在血细胞、外套膜和鳃中。在鳗弧菌(Vibrio anguillarum)刺激后,血细胞中的hdpgrp表达量在72 h内呈现先上升后下降的趋势,在24 h表达量达到最高。SDS-PAGE结果显示,重组HdPGRP (rHdPGRP)的分子量为30 kDa。rHdPGRP表现为Zn2+依赖酰胺酶活性,可催化降解不溶性肽聚糖。此外,rHdPGRP对革兰氏阳性菌藤黄微球菌(Micrococcus luteus)具有显著的抑制作用,且这种抑制作用可能与其酰胺酶活性有关。本研究表明,HdPGRP在机体抵御入侵细菌等免疫防御中起重要作用。     

Bioassay‐guided isolation and identification of active compounds from Macleaya microcarpa (Maxim) Fedde against fish pathogenic bacteria

Four alkaloids (Sanguinarine, 6‐Methoxyl‐dihydro‐chelerythrine, Cryptopine and β‐Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay‐guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram‐negative strains than other three alkaloids, with MIC values of 12.5 mg L^?1 for A. hydrophila and 50 mg L^?1 to other pathogenic bacteria. Followed by 6‐methoxyl‐dihydro‐chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L^?1 for A. hydrophila, 100 mg L^?1 for V. harveyi, and 125 mg L^?1 for both V. anguillarum and A. salmonicida. Cryptopine and β‐allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L^?1 for A. hydrophila and 200 mg L^?1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.     

Newly identified C‐type lysozyme in Chinese soft‐shelled turtle (Pelodiscus sinensis) exhibits potent antimicrobial activity

Lysozymes play vital roles in humoural immune response against bacterial invasion by its lytic activity. In the present study, a new C‐type lysozyme was identified and characterized from Chinese soft‐shelled turtle Pelodiscus sinensis. The full‐length cDNA of PslysC was of 923 bp, encoding a polypeptide of 148 amino acid residues. The multiple alignments and phylogenetic relationship analysis revealed the highly enzyme‐related conserved residues. The real‐time PCR analysis suggested that PslysC was constitutively expressed in a wide range of tissues with highest level in blood cells and liver. The expression of PslysC could be significantly up‐regulated under Aeromonas jandaei infection and ammonia exposure, while no significant changes were found under Poly I:C infection. The rPslysC protein was expressed in E. coli and purified by Ni‐NTA. The optimal pH and temperature for rPslysC protein lytic activities were determined at pH 7 and 30℃. rPslysC can inhibit the growth of eight kinds of Gram‐negative bacteria, and three kinds of Gram‐positive bacteria. The binding activity of rPslysC to different microbial polysaccharides and microorganism was analysed. The results showed that rPslysC could bind to selected bacteria, and exhibit a strong binding activity to lipopolysaccharide and peptidoglycan, but a weak binding activity to β‐glucan. This suggests that the binding activity might be the major mechanism of action to realize the antibacterial activity. The present study will provide helpful evidence to further understand the innate immunity of P. sinensis, and the interaction mechanisms of C‐type lysozymes with bacterial membranes.     

对虾养殖池中一株弧菌拮抗菌的分离鉴定

从对虾养殖池中分离出1株编号为2013082515(简称菌株15)的菌株,以鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(Vibrio harveyi)和副溶血弧菌(Vibrio parahaemolyticus)为指示菌,分析了菌株15对指示菌的抑菌效果及最低抑菌浓度,同时,分析了菌株15对其他7株弧菌的抑菌效果.结果显示,菌株15对3株指示菌均具有抑菌效果,对鳗弧菌、哈维氏弧菌及副溶血弧菌的最低抑菌浓度分别为2.50×104、2.50×105、2.50×105 CFU/ml;对其他弧菌也具有一定的抑菌效果.采用注射及浸泡感染方法分析了菌株15对对虾的生物安全性,结果显示,在高浓度时,菌株15对对虾具有潜在毒性.分别用细菌全细胞脂肪酸气相色谱法和16S rDNA序列分析比对法对该菌进行分类鉴定,表明菌株15为一株假交替单胞菌(Pseudoalteromonas sp.).     

拟态弧菌感染黄颡鱼的动态病理损伤及病原分布研究

以1.0×106CFU·m L~(-1)拟态弧菌(Vibrio mimicus)浸浴感染黄颡鱼(Pelteobagrus fulvidraco),于第0、第4、第8、第16、第24、第36、第48、第60、第72小时剖杀取样,研究病理损伤及病原菌的体内动态分布。结果显示,皮肤肌肉、鳃、肠道出现病变最早,其中皮肤肌肉损伤最严重。第16小时表皮变性、坏死,鳃上皮肿胀,肠绒毛水肿;第24~第48小时表皮坏死、脱落,真皮严重出血,肌纤维变性、坏死,鳃出血,上皮局部坏死,肠上皮变性与灶性坏死;第48~第72小时皮肤肌肉坏死更严重,形成溃疡灶,鳃灶性坏死。肾、肝、脾与心36 h后相继出现不同程度的淤血、出血,变性和灶性坏死。q PCR检测发现,第4小时即在鳃、肠、皮肤肌肉检出病菌,细菌含量分别为1.3×102CFU·mg~(-1)、2.6×102CFU·mg~(-1)、4.7×102CFU·mg~(-1);鳃与皮肤肌肉中菌量随时间延长而增加,第72小时皮肤肌肉中菌量达到4.5×107CFU·mg~(-1);第24小时后相继在肾、肝、脾、心检出病菌,菌量介于1.9×10~4.7×102CFU·mg~(-1)之间。结果表明皮肤、鳃和肠道是病菌的入侵位点,并在体内多组织、器官分布。     

Antibacterial abilities of intestinal bacteria from larval and juvenile Japanese flounder against fish pathogens

The present study was undertaken to examine the antibacterial abilities of intestinal bacteria isolated from juveniles and larvae of Japanese flounder Paralichthys olivaceus . Newly hatched larvae of flounder were held in a 100 L plastic circular tank and fed rotifiers, Artemia nauplii and commercial feeds, depending on the developmental stage of the fish. Genera Aeromonas , Moraxella and Vibrio were predominantly isolated from the intestinal tracts of Japanese flounder at larval and juvenile stages, whereas Aeromonas , Bacillus , coryneforms, Moraxella , Pseudomonas and Vibrio were detected at high densities in live diets and artificial feeds. Antibacterial bacteria accounted for 1.7–24.3% of the intestinal isolates against Lactococcus garvieae , Pasteurella piscicida , Vibrio anguillarum and V. vulnificus . In particular, as much as 53.3% of Vibrio spp. other than Vibrio -swarmer isolated from 197-day-old juveniles inhibited the growth of P. piscicida . These results suggest that intestinal bacteria having antibacterial activity play a role in the prevention of infectious diseases.