牙鲆抗菌肽hepcidin基因在成鱼组织和不同发育时期胚胎中的表达分析

抗菌肽hepcidin是由hepcidin基因编码产生的小分子多肽,是机体天然免疫的重要因子。本文利用RT-PCR的方法分析表明,抗菌肽hepcidin基因在牙鲆成鱼不同组织和不同发育时期胚胎中普遍表达,但是表达水平存在着明显的差异。     

爱德华氏菌灭活疫苗对黄颡鱼免疫相关基因表达及过氧化氢酶、超氧化歧化酶和补体C3活性的影响

为评价爱德华氏菌灭活疫苗对黄颡鱼的免疫效果,分析了灭活爱德华氏菌疫苗对黄颡鱼非特异性免疫应答相关的免疫基因表达和酶活性的影响。结果显示,黄颡鱼鳃组织中抗菌肽基因和肝组织中长型肽聚糖识别蛋白基因在免疫7、14、21、28d后的表达均显著上调,肠道中抗菌肽基因和脾脏组织中长型肽聚糖识别蛋白基因的表达在免疫7、14、21d亦显著上升。血清中过氧化氢酶、超氧化歧化酶和补体C3活性出现不同程度的提高,其中,黄颡鱼过氧化氢酶活力在免疫14、21d后显著高于对照组,超氧化歧化酶活力在免疫14、21、28d后显著高于对照组,在21d达到峰值,补体C3在免疫7、14、21、28d后均显著上升(P0.05),在免疫7d后血清中补体C3的含量达到高峰。试验结果表明,爱德华氏菌灭活疫苗可显著提高黄颡鱼免疫基因抗菌肽和长型肽聚糖识别蛋白的表达以及酶的活性,进一步揭示爱德华氏菌灭活疫苗可提高黄颡鱼的非特异性免疫能力。     

以莱茵衣藻为载体表达外源抗菌肽的研究

将黑水虻抗菌肽基因sarcotoxin3转入到莱茵衣藻细胞中,以期实现抗菌肽活性片段的大规模表达生产,为将来完成抗菌肽的临床测试及水产应用奠定实验基础。以质粒pHK85为骨架,插入荧光素酶基因和黑水虻抗菌肽sarcotoxin3基因,用DNA连接酶将质粒连接,构成抗菌肽质粒。抗菌肽质粒经克隆、提取、酶切线性化、衣藻玻璃珠转化、荧光素酶活性筛选和抑菌实验,结果表明:经过测序进一步确认质粒成功构建,碱基序列与所设计质粒序列一致,质粒浓度472.8 ng/μL、纯度(A_(260)/A_(280))1.93,7 d后每个巴龙霉素平板长有单克隆约200个,大多数单克隆荧光素酶活力值可高达10~6,少数单克隆荧光值甚至可达10~8;藻株在26 kDa附近出现了特异信号,含有抗菌肽的蛋白对大肠杆菌DH5α有一定的抑制作用。莱茵衣藻可以作为抗菌肽外源表达的一种载体,为抗菌肽的大量生产提供了一种可能。     

传统抗生素替代物抗菌肽在水产养殖中的应用前景

抗菌肽(antibacterial peptides)是具有抗菌活性基因编码的一类短肽,目前发现抗菌肽或类似抗菌肽的小分子肽类广泛存在于昆虫、两栖类、水产动物及哺乳动物中,这种内源性抗菌肽经诱导而合成,在机体抵抗病原的入侵方面起着重要的作用,更被认为是缺乏特异性免疫功能生物的重要防御成分.该文系统介绍了抗菌肽的定义、结构、作用机理、与传统抗生素的差异以及在水产养殖中的应用前景,以期为抗菌肽进一步深入研究提供参考.     

嗜水气单胞菌感染对黄鳝hepcidin抗菌肽基因表达的影响

为了研究嗜水气单胞菌对黄鳝hepcidin抗菌肽基因表达的影响,用不同密度嗜水气单胞菌感染健康黄鳝后,采用荧光定量PCR方法分别检测染菌黄鳝肾脏hepcidin抗菌肽基因在感染后24h、48h、72h的表达量。试验结果表明,hepcidin基因在健康黄鳝中就有表达,腹腔注射嗜水气单胞菌后,该基因的表达量明显升高。随着处理时间的延长,处理菌密度较低组(0.5×108 cfu/mL)和较高组(2×108 cfu/mL和4×108 cfu/mL)黄鳝抗菌肽hepcidin基因的表达量都逐渐降低,只有菌密度为1×108cfu/mL时该基因的表达量是随着处理时间的延长而逐渐升高,但升高的幅度逐渐减小。密度为1×108 cfu/mL的嗜水气单胞菌能诱导黄鳝hepcidin抗菌肽基因表达量逐渐增加。     

水产动物基因工程抗菌肽研究及其应用前景的研究

抗菌肽是水产动物体内非特异性免疫系统的第一道防线,在抵御外来病原微生物的过程中发挥着重要作用.来源于鱼、虾、蟹等水产动物的抗菌肽杀菌作用更强,溶血效应小,种类多样化,显示出比其它种类更具优势的应用前景.介绍了一些水产动物抗菌肽的研究近况、基因工程研究进展以及转抗菌肽基因在水产动物中的研究进展,并展望了其在水产领域的应用前景.     

海洋生物鲎抗菌肽研究进展

抗菌肽是基因编码的肽类抗菌分子,广泛分布于整个生物界,是脊椎动物、无脊椎动物和植物先天免疫的关键因子.本文综述了海洋动物鲎目前抗菌肽的种类、结构、功能及分子生物学等研究状况.     

斑点叉尾鲖BPI1基因的原核表达及生物信息学分析

为探究斑点叉尾鲖BPI1抗菌肽基因编码蛋白的结构特征,实验提取斑点叉尾鲖肾脏RNA,根据BPI1基因序列设计引物,利用PCR方法克隆BPI1基因并构建至原核表达载体p TWIN1中。构建成功的重组质粒p TWIN1-BPI1经原核表达,SDS-PAGE检测显示得到约为50 ku的融合蛋白,与预期结果相一致。通过生物信息学软件对BPI1基因编码的蛋白序列进行分析,结果显示,克隆所得斑点叉尾鲖BPI1基因编码一条由223个氨基酸残基组成的多肽,等电点为9.07,属于BPI超家族,是稳定的亲水蛋白。亚细胞定位BPI1分布在线粒体(43.5%)、细胞质(30.4%)和细胞核(26.1%)中,表明BPI1可能在嘌呤和嘧啶合成以及能量代谢等过程中发挥信号转导、促进生长等重要作用。二级结构以α-螺旋和β-折叠为主,三级结构呈棒状。同源性及进化树分析表明,实验所得BPI1基因序列与斑点叉尾鲖BPI抗菌肽基因的同源性最高,序列一致性为99.1%,进化树聚为一支,表明BPI1基因编码的蛋白序列在相同物种中的突变率较低,保守性较高,相同物种中其生物活性与个体间的差异关系较小。     

背角无齿蚌抗菌肽theromacin基因cDNA全序列克隆及表达分析

采用RACE方法,克隆了背角无齿蚌抗菌肽theromacin基因的cDNA全序列。结果显示,该cDNA序列全长为870 bp,5'端非翻译区104 bp,3'端非翻译区460 bp,开放阅读框长为306 bp,共编码101个氨基酸,相对分子量为11 166.9 u。同源性分析显示,该基因编码的蛋白与三角帆蚌theromacin基因氨基酸序列的相似度最高,为73%;与贻贝中发现的抗菌肽defensins、mytilins、myticins和mytimycins等4种类型相似度较低,属于Macin家族(登录号:KJ598604)。实时荧光定量PCR分析结果显示,该基因在血液、肝脏、外套膜、鳃、斧足和肠等组织中均有表达,在外套膜中的表达量最高,在其他组织中的表达量较低;经嗜水气单胞菌诱导后,各个组织的表达量出现明显上调的趋势且与对照组显著差异,推测theromacin基因可能在背角无齿蚌的免疫反应中起重要作用。     

三角帆蚌外套膜表达的免疫相关基因筛选

利用三角帆蚌(Hyriopsis cumingii)外套膜cDNA文库EST序列,通过BLAST分析注释基因功能,发现35个与免疫防御功能相关的基因。根据其功能,这些免疫相关基因可划分为7类,即细胞免疫过程(2个)、蛋白酶和蛋白酶调节子(9个)、压力蛋白(7个)、抗菌肽(5个)、溶酶体酶(2个)、粘着蛋白(3个)及细胞凋亡和细胞周期调控相关基因(7个)。免疫相关基因在三角帆蚌外套膜中的广泛表达表明外套膜是重要的免疫组织。该研究为三角帆蚌外套膜分子免疫机理研究和抗病育种工作提供了基础性资料。     

Estimation of variation of virulence of Renibacterium salmoninarum by survival analysis of experimental infection of salmonid fish

The variation of virulence of Renibacterium salmoninarum , the causative agent of bacterial kidney disease (BKD) in salmonid fish, was studied by infecting rainbow trout, Oncorhynchus mykiss (Walbaum), with two isolates (strains 325 and 932) from diseased Atlantic salmon, Salmo salar L., and one isolate (strain 4366) from an apparently healthy Atlantic salmon. Coho salmon, Oncorhynchus kisutch (Walbaum), were injected with the strain 932 to estimate difference in fish species resistance. Fish were removed by random sampling for other study purposes, a study design possible with analysis of lifetime distributions incorporating both sampling-, death- and survival-times. At the end of the experiment, the rainbow trout infected with strains 325, 932 and 4366 had a survival probability of 33%, 51% and 72%, respectively. The coho salmon infected with strain 932 had a 26% survival probability. The strain differences were significant according to the log-rank test, and the risk ratio between the strains ranged from 1·8 to 5·4. The strain from the apparently healthy fish was least virulent. The survival of the fish species was different over time. Rainbow trout were more likely to die early in the time course, but high numbers of coho died later, resulting in an overall risk of mortality of 1·4 in favour of rainbow trout. Differences in virulence may reflect changed selective pressure on R. salmoninarum when introduced from feral stocks into the environment of fish farms.     

母猪胎衣不下的病因与防治要点

猪的胎盘属于弥散型胎盘,这种胎盘的结构特点和饲养管理的不当,常常导致母猪胎衣不下发生,给生猪的生产繁殖带来极大损失。本文针对母猪胎衣不下发生病因、综合防治进行详细阐述,旨在对预防和治疗胎衣不下能有所帮助。     

水硬度对七彩神仙鱼幼鱼发育的影响

采用不同硬度的水对七彩神仙鱼幼鱼进行饲养。6周龄幼鱼在硬度为7.94°dH±0.30°dH时饲养84d后,比在硬度为14.71°dH±0.23°dH水中的幼鱼个体大,生长速度快。表明较高硬度的水有利于七彩神仙鱼幼鱼的生长发育。     

虎斑乌贼受精卵卵黄营养成分分析

本实验对虎斑乌贼受精卵卵黄的营养成分进行分析,旨在探讨其幼体的营养需求量,为其幼体配合饲料研制提供参考数据。随机选取大约800个虎斑乌贼受精卵的卵黄,采用国家标准方法测定其水分、灰分、粗蛋白质、粗脂肪、氨基酸、脂肪酸和矿物元素含量。结果表明:1)虎斑乌贼受精卵卵黄中粗蛋白质含量为76.33%(干重基础);总氨基酸(TAA)和必需氨基酸(EAA)含量分别为71.22%和32.38%(干重基础),EAA/TAA为45.46%,氨基酸中以谷氨酸(Glu)含量最高(9.97%),必需氨基酸中亮氨酸(Leu)含量最高(7.58%)。2)其粗脂肪含量12.71%(干重基础);共检出17种脂肪酸,包括8种饱和脂肪酸(SFA)、5种单不饱和脂肪酸(MUFA)和4种多不饱和脂肪酸(PUFA),SFA、MUFA和PUFA分别占脂肪酸总量的43.47%、7.54%和49.25%,其中以DHA含量最高,达32.80%,EPA含量为7.70%,DHA/EPA为4.26。3)检测出Na、K、Ca、Mg、Sr、Mn、Fe、Cu、Zn、Al和As 矿物元素,微量元素中富含Zn、Al和Fe,含量分别为 0.77、0.71和0.43 mg/kg(鲜重基础)。由此可见,卵黄具有高蛋白、低脂肪,富含n-3PUFA的特点;虎斑乌贼幼体饲料中蛋白质需求量参考值为76.33%;氨基酸需求量参考值,如赖氨酸(Lys)为5.49%,蛋氨酸(Met)为2.63%;脂肪的需求量参考值为12.71%,DHA为4.17%,EPA为0.98%;微量元素需求量参考值,如Zn为2.77 mg/kg,Cu为0.19 mg/kg(干重基础)。     

斑节对虾虾苗活力检测方法

该研究通过肉眼观察、镜检，进行干露、饥饿、盐度突降、福尔马林等抗性试验，并采用病毒检测等方法，以期建立评估斑节对虾（Penaeus monodon）虾苗活力和质量标准。结果表明，斑节对虾健康虾苗具有趋光性、集群性，体表光洁，肌肉透亮，肠胃食物充盈等特性。测试虾苗干露时间以15min为宜，健康虾苗干露后能立即恢复活力，而病弱虾苗多出现死亡、昏迷现象；虾苗的成活率随饥饿时间的延长而降低，随福尔马林浓度升高和时间延长而降低，随盐度突降幅度增加而降低。健康虾苗能忍受100～200μL·L^-1福尔马林溶液30min，成活率近100％；在盐度20～30下虾苗的成活情况较好，而其在淡水中仅能存活1h。对虾苗进行病毒检测，可以避免养殖中因虾苗携带病毒而可能导致的病毒性疾病的暴发。     

金鳟、虹鳟苗种长途运输实例

鳟鱼发眼卵和旨种运输是养鳟生产的重要环节。发眼卵多在冬季和早春运输，苗种多在晚春和夏季运输。苗种运输大多以鱼篓充氧进行短距离汽车运输，长途运输冷水性鱼苗种的实例很少。本文总结了金鳟和虹鳟苗种长途运输方法，以期与养鳟业界共同交流提高。一、包装材料和方法内包装材料是苗种运输专用塑料袋，规格54×104cm，外包装是泡沫塑料箱，规格63.5×45.5×30．5cm，纸箱规格64×46×32．5cm。塑料袋采取双层式，内装苗种和水共12.5kg，其中苗种0.5-1．3kg，100－2000尾。起运前苗种停食1－2天。运鱼用水水温调至6℃，鱼、水入袋后袋内…     

Characterization of protease inhibitors of seminal plasma of cyprinids

Anti-proteinase activity was demonstrated in the seminal plasma of cyprinid fish species (bream, chub, ide, dace, asp, goldfish, roach, common carp) using electrophoretic techniques combined with a detection method based on inhibition of bovine trypsin. We found species-specific protease inhibitors in the seminal plasma of cyprinids. At least three bands of protease inhibitors with different migration rates could be identified by native PAGE. Higher variability was characterized for bands with slower migration rates. Visualization of inhibitors after SDS-PAGE under non-reducing conditions allowed estimation of their molecular weights. Apparent molecular weights were within the range of 51–59 and 47–54 kDa for the bands with slower and moderate migration rates, respectively. The molecular weight of fast migration bands for roach and common carp were estimated to 23 and 30 kDa, respectively. Inhibitors of common carp seminal plasma differed in their affinity toward serine proteases. Three inhibitors in common carp seminal plasma could be visualized using cod and bovine trypsin, but only two inhibitors (of high molecular weight) were recognized with chymotrypsin. There were differences in anti-proteinase activity and seminal plasma protein concentration in relation to the origin of common carp seminal plasma (breeding lines) and time of milt collection (spawning vs. post-spawning season).     

鱼类热忍耐温度研究进展

The paper is a review of the studies on the temperature of the thermal tolerance in fishes. It was described from four aspect: (1) The key parameters of temperature of thermal tolerance in fishes; (2) The influence factors of temperature of thermal tolerance in fishes; (3) The research methods of thermal effect of fishes; (4) The evaluation of effects of thermal pollution on fishes, especially, with the coastal power station will be‘built in China recent years, the effects of thermal discharge water on the marine fisheries resource wered iscussed.     

Histopathology of respiratory organs of certain air-breathing fishes of India

Air-breathing fishes have evolved bimodal respiratory mechanisms for exploitation of water (through gills and highly vascularized skin) as well as atmospheric air (through aerial respiratory organs, ABO). Mucous cells in these respiratory organs of variously stressed fishes exhibit periodic fluctuations in their density and staining properties. The main types of damage in the gills include congestion of blood capillaries (BLCs), periodic lifting and sloughing of respiratory epithelia of the secondary lamellae causing haemorrhage, extensive fusion of secondary lamellae and hyperplasia of the respiratory epithelia due to uncontrolled regeneration leading to asphyxiation, altered excretion, and death of the fish. Haemolysis has also been observed following lead exposure. The damage in the ABO of Heteropneustes fossilis includes sloughing of the epithelial cells, leading to haemorrhage causing decreased red blood corpuscles density and degeneration of the secondary gill lamellae with reduced respiratory area. Subsequent hyperplasia of the respiratory epithelia and fusion of gill lamellae increase the respiratory barrier distance. The BLCs often bulge out and protrude into the lumen, bringing blood nearer to air. The ladder-like pillar cell (PLC)-BLC components of the gill lamellae frequently collapse. Damage to the ABO of Channa striata is less severe. Often haemorrhaging due to bursting of extensively stretched BLCs causes aerial respiratory failure. Chloride cells of the ABOs also show hyperplasia. While the highly mucogenic epidermis of C. striata shows less damage, the epidermis of Clarias batrachus and H. fossilis shows severe wear and tear, sloughing, and haemorrhage. Side-by-side regeneration continues, causing altered histomorphology of the epidermis. The different gland cells also show periodic fluctuations in their density and staining. The dermis also shows severe damage with loosening of their connective tissue fibres. These fibres give stronger reactions for sulfated mucin that not only retain additional water molecules for continuance of skin breathing, but also bind the toxic ambient pollutants.     

牙鲆刺激隐核虫病的防治

2005年七、八月份,乐亭、滦南一带多家工厂化牙鲆养殖场发生刺激隐核虫病,此种病虫害发病急、传染快、死亡率高。发生过该病的养殖场牙鲆死亡率一般都在50％～80％之间,这种病害给牙鲆养殖业带来很大损失。