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。     

大口黑鲈hepcidin真核表达载体构建及表达的初步研究

将大口黑鲈(Micropterus salmoides)抗菌肽hepcidin cDNA定向插入真核表达载体pPICZαA,通过SacI酶切线性化重组表达质粒pPICZαA-hepcidin,电转化毕赤酵母P.pastoris GS115,PCR扩增筛选,甲醇诱导表达等进行了hepcidin真核表达工程菌株的构建及诱导表达。结果显示:hepcidin cDNA成功插入表达载体pPICZαA,并整合到酵母基因组中;经甲醇诱导,RT-PCR检测显示hepcidin cDNA在酵母细胞中成功转录。     

鳜hepcidin cDNA的分子克隆及序列分析

Hepcidin是近年来发现的一类具有独特性质的抗菌肽，根据C-enBank中收录的鱼类抗菌肽hepcidin的cDNA序列设计一对简并引物，用RT-PCR方法从鳜（Sinipercachuatsi）肝脏组织中克隆到hepcidin的cDNA，并构建pMD18-T-hep-cidin载体进行序列测定和分析。结果表明，该cDNA长为381bp，其中第20-277位是该基因的开放阅读框（ORF），编码86个氨基酸，形成由信号肽（24个残基）、前肽（42个残基）和成熟肽（20个残基）3部分序列组成的前体肽，与已报道的其他鲈形目鱼类hepcidin相比较，核苷酸序列的同源性为72．2％-92．0％，所推导的成熟肽与包括人类在内的其他生物hepcidin成熟肽的同源性在50％-86．4％，都含有8个保守的cys残基，可形成4个链内二硫键。鳜hepcidin cDNA片段的获得为以后的重组表达奠定了实验基础，也为抗菌肽hepcidin家族找到了新的成员。     

鱼类抗菌肽研究进展（二）

<正>3 Hepcidin Hepcidin是一种富含半胱氨酸的多肽,最先在人源的hepcidin中发现其具有抗菌活性。此后,hepcidin在许多其他的脊椎动物被陆续发现,包括爬行类,两栖类和鱼类。在鱼类中,hepcidin最先从杂交条纹鲈中发现,目前至少已经在37种鱼类中鉴定到了该抗菌肽[1]。Hepcidin一般是一个由4个二硫桥形成harpin-折叠,包含8个半胱氨酸,然而鱼hepcidin序列分析表明,鱼类hepcidin只含有4,6或7个半胱氨酸[2]。鱼类hepcidin基因在进化过程中发生了重复和多样化的过程中,产生了多重基因拷贝的,最多     

斜带石斑鱼抗菌肽hepcidin基因克隆及其成熟肽的原核融合表达

文章根据已报道的硬骨鱼类hepcidin cDNA序列设计简并引物，通过RT—PCR从重要海水经济鱼类斜带石斑鱼（Epinephelus coioides）肝脏中扩增出1条具有完整开放阅读框的246bpcDNA序列，Blast分析表明该序列是鱼类hepcidin基因家族的一员，被命名为斜带石斑鱼hepcidin样抗菌肽前体，该cDNA所推导的氨基酸序列有如下特点：1）信号肽序列与多数鱼类hepcidin信号肽的同源性在67％～87％之间；2）C端20个氨基酸序列具有绝大多数动物hepcidin成熟肽的共同典型保守序列特征，即在对应位置具有8个Cys；3）序列中不具有已报道的hepcidin前体肽转化酶作用典型基序[RX（K／R）R]；4）与GenBank中已注册的2条斜带石斑鱼hepcidincDNA序列（GU391241和GU391242）所推导的氨基酸序列同源性分别为36％和33％，且NJ进化树显示不与这2条序列聚为一枝，表明该序列是斜带石斑鱼hepcidin基因家族中一种新亚型，其预测成熟肽融合表达载体成功在大肠杆菌（Escherichiacoli）中表达，为后续研究奠定基础。     

真鲷鳃组织cDNA文库的构建与hepcidin抗菌肽基因序列的扩增

以细菌攻毒的真鲷鳃组织为材料，分离出mRNA，合成双链后，回收500～4 000 bp cDNA片段，构建了λZAP表达型cDNA文库。初级文库的容量为1.75 ×105个重组子，扩增文库的滴度达到1×10⁹ pfu·mL^-1，随机挑选噬菌斑的插入片断在500～2000 bp之间。以hepcidin特异性引物做PCR扩增文库，获得了hepcidin抗菌肽基因cDNA序列，证明所建立的文库可用于免疫相关基因的筛选。本文库可作为筛选真鲷免疫相关基因的重要资源。     

水生动物抗菌肽(Antibacterial Peptides)的研究概况

简要介绍了国内外水生动物抗菌肽的研究概况，包括其理化性质、生物活性及利用基因工程技术克隆和表达抗菌肽基因等，并提出抗菌肽在水产养殖中的应用前景。     

中国明对虾抗菌肽基因应答WSSV侵染的表达及其SNP分析

通过白斑综合征病毒(WSSV)感染实验,利用实时定量PCR技术研究了中国明对虾(Fenneropenaeus chinensis)应答病毒侵染后,已知的3种抗菌肽(对虾肽)在肝胰腺、肌肉、肠和鳃4种组织中的差异表达情况.结果显示,虽然3种抗菌肽表现出明显的组织表达特异性,即在不同组织中的表达趋势和表达丰富度存在明显的差异,但是就同一个组织而言,3种抗菌肽在1～120 h WSSV侵染区间内的表达趋势基本一致,在0 h(未侵染病毒)时,3种抗菌肽的表达量极低(为0);在6～24 h期间,检测到明显的表达量;48～120 h期间,3种抗菌肽的表达量总体呈现下降的趋势.这暗示3种抗菌肽在对虾机体内可能具有相似的生物学功能.在此基础上,本研究对各类型中国明对虾抗菌肽的SNP位点进行了筛选,进一步对不同SNP类型与抗WSSV或易感WSSV的关联程度进行了分析,结果显示3种抗菌肽基因的SNP位点很少,且在抗性和易感对虾群体内不存在明显的偏向分布.     

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。     

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。     

Effect of dietary iron levels on growth,iron concentration in tissues,and blood concentration levels of transferrin and hepcidin in bighead carp (Aristichthys nobilis)

A 60‐day feeding trial was conducted to estimate the effects of dietary iron (Fe) levels on growth, Fe concentration in the liver, spleen, and blood, and transferrin and hepcidin concentrations in the blood of bighead carp (Aristichthys nobilis). The six experimental diets were formulated to contain different Fe levels (0, 43.1, 84.2, 123.3, 162.2 and 203.1 mg/kg of dry diet) using ferrous sulphate (FeSO₄) as the source. The weight gain (WG) and the specific growth ratio (SGR) of A. nobilis fed with a dietary Fe level of 123.3 mg/kg were significantly higher than that of the 0 mg/kg Fe group (p < .05). The results indicated that the growth was affected by dietary Fe levels. Regression analysis of WG and SGR at different levels of dietary Fe suggests that the appropriate dietary requirement of Fe for the bighead carp larvae is 120–134.36 mg/kg. The Fe contents in different tissues were as follows: spleen > liver > whole body. When the Fe dietary content increases to 162.2 mg/kg, the blood concentrations of Fe significantly decreased and thereafter increased, hepcidin significantly decreased and thereafter decreased, and transferrin significantly increased and thereafter decreased. The results indicate that the transferrin blood content significantly increased with decreasing hepcidin of up to 264.63 μg/ml content and thereafter decreased. It could be concluded that after transferrin saturation, hepcidin functions to maintain iron balance in the blood of A. nobilis by decreasing transferrin content.     

Immunomodulation of rainbow trout (Oncorhynchus mykiss) fry by bath exposure to a β‐glucan from Euglena gracilis

Early developmental stages of fish mostly depend on innate immune factors for their protection. Augmenting these factors by application of different immunostimulatory substances may be beneficial for rearing and survival of the early life stages of fish. Bath administration of stimulants leads to a uniform exposure of fish independent of feed intake and reduces the individual handling. The present study demonstrates the immunostimulatory effect of β‐glucan (bath exposure) in rainbow trout fry at different dosages and exposure time. Rainbow trout fry (avg. wt. 770 mg; 87 days post hatch) were exposed to three different concentrations of β‐glucan (10, 100 and 1000 μg mL^?1) by bath exposure for 1 and 24 h. Expression of immune related genes from pooled internal organ samples of individual fish were analysed using a real time qPCR assay. Expression of complement factors (C3 and factor B) and acute phase proteins (hepcidin, precerebellin and transferrin) was significantly up‐regulated after 24 h bath stimulation with β‐glucan (100 μg mL^?1). These innate immune factors may play a vital role in clearance of pathogens. The expression of most of genes showed both a dose‐ and time‐dependent response. A medium dose (100 μg mL^?1) induced a significant increase in expression of complement factors and acute phase proteins mainly at 24 h exposure, whereas the highest dose of β‐glucan (1000 μg mL^?1) down‐regulated the expression of most of the studied genes. The result from the present study indicates that β‐glucan bath exposure could be applied for enhancing the innate immune factors even in fry.     

急性胁迫对团头鲂铁稳态及其相关基因表达的影响

为研究急性应激反应对鱼体铁含量及铁稳态相关基因的影响,以团头鲂为研究对象,通过腹腔注射皮质醇来模拟急性应激,在注射后不同时间点对团头鲂的血液、肠和肝脏进行取样,采用分光光度法测定血清和肝脏中的铁含量,采用实时荧光定量PCR对铁稳态相关基因的表达量进行检测。结果显示,皮质醇注射后血浆皮质醇显著升高,血浆中铁含量在4、8、10和12 h显著高于对照组,而肝脏中的铁含量在4、8、10和12 h时显著低于对照组。团头鲂肝脏中铁调素(hep)基因表达量显著增加,并分别在8和10 h达最大值,随后有所下降。肠道和肝脏中转铁蛋白基因(Tf)的表达量均呈先升高后下降的趋势。研究表明,急性应激下机体细胞内储存铁大量释放到体液中,造成病原感染和体内增殖风险增加;hep及Tf上调表达降低细胞内铁释放和促进体液铁向胞内蓄积,在鱼体内铁稳态的调控和固有免疫反应中发挥着重要的作用。     

低温驯化下4种不同品系罗非鱼血清皮质醇与免疫相关指标的变化

选取4种不同品系罗非鱼, 分别为吉富罗非鱼(Oreochromis niloticus)、奥尼罗非鱼(O. niloticus×O. aureus)、红罗非鱼(O. nilotica♂× O. mossambica♀)和奥利亚罗非鱼(O. aureaus)。在26℃水温下饲养3周后, 选取规格基本一致的罗非鱼(体质量50.73 g±4.23 g)进行低温驯化实验。水温以3℃/d的速度从26℃降至8℃, 分别于水温为26℃、20℃、14℃和8℃时进行采样, 比较不同驯化阶段4种不同品系罗非鱼血清皮质醇和免疫相关指标的变化规律。结果表明, 与26℃时的免疫指标相比, 水温降至8℃时, 吉富与红罗非鱼血清皮质醇水平显著升高; 然而血清C3、C4、IgM以及头肾C型溶菌酶mRNA水平显著下降(P<0.05), 血清中高皮质醇水平对鱼体的免疫产生了抑制作用。水温为8℃时, 奥尼罗非鱼血清皮质醇、IgM和补体C3以及头肾抗菌肽mRNA水平显著升高; 奥利亚血清皮质醇、C4和IgM以及头肾C型溶菌酶mRNA水平与26℃时相比无显著差异, 然而血清溶菌酶与C3水平降低。驯化实验结束后, 比较了4种不同品系罗非鱼在8℃水温下48 h内的累积死亡率。吉富与红罗非鱼组累积死亡率较高, 分别达到43.3%和40.0%; 奥尼罗非鱼其次, 为23.3%; 奥利亚罗非鱼最低, 为20.0%。较高的血清IgM和头肾溶菌酶和抗菌肽mRNA水平可能有助于提高奥尼和奥利亚罗非鱼的抗低温应激能力, 增加低温时的成活率。本研究通过分析4种品系罗非鱼不同驯化阶段血清皮质醇和免疫相关指标, 探讨不同品系罗非鱼在低温驯化过程中的免疫保护机制, 旨在为下一步抗低温新品系罗非鱼的选育提供理论依据。

     

Immunostimulatory effects of dietary poly‐β‐hydroxybutyrate in European sea bass postlarvae

The stable production of high‐quality fry in marine aquaculture is still hampered by unpredictable mortality caused by infectious diseases during larval rearing. Consequently, the development of new biocontrol agents is crucial for a viable aquaculture industry. The bacterial energy storage compound poly‐β‐hydroxybutyrate (PHB) has been shown to exhibit beneficial properties on aquatic organisms such as enhanced survival, growth, disease resistance and a controlling effect on the gastrointestinal microbiota. However, the effect of PHB on the developing immune system of fish larvae has so far not been investigated. In this study, the effect of feeding PHB‐enriched Artemia nauplii on survival, growth and immune response in European sea bass (Dicentrarchus labrax) postlarvae was examined. Amorphous PHB was administered to 28‐day‐old sea bass postlarvae over a period of 10 days. The survival and growth performance were monitored, and the expression of 29 genes involved in immunity, growth, metabolism and stress‐response was measured. While the expression of the insulin‐like growth factor 1 (igf1), an indicator of relative growth, was upregulated in response to feeding PHB, the larval survival and growth performance remained unaffected. After 10 days of PHB treatment, the expression of the antimicrobial peptides dicentracin (dic) and hepcidin (hep) as well as mhc class IIa and mhc class IIb was elevated in the PHB fed postlarvae. This indicates that PHB is capable of stimulating the immune system of fish early life stages, which may be the cause of the increased resistance to diseases and robustness observed in previous studies.     

In vivo screening of zebrafish microRNA responses to bacterial infection and their possible roles in regulating immune response genes after lipopolysaccharide stimulation

Micro (mi)RNAs are abundant small noncoding RNAs found in plants and animals, the regulatory functions of which are not fully understood in fish. To identify potential miRNAs, we screened an miRNA microarray with total RNA from zebrafish infected with Vibrio harveyi and another from uninfected zebrafish. Six miRNAs were obtained from the microarray screening. We studied miRNA expression patterns of 2 miRNAs (miR-122 and miR-194) after bacterial infection of transgenic zebrafish (containing tilapia hepcidin (TH)2-3) and non-transgenic zebrafish from which the 2 miRNAs were obtained from the microarray experiment. The results indicated that miR-122 and miR-194 were higher in PBS-injected zebrafish compared with TH2-3 zebrafish or wild-type (WT) zebrafish after V. harveyi infection. Overexpression of miRNAs (miR-122, miR-192, and miR-194a) was seen in zebrafish liver (ZFL) cells after lipopolysaccharide (LPS) treatment and in untreated fish. Our results showed that after 24?h of doxycycline treatment without LPS stimulation, interleukin (IL)-22, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, and tumor necrosis factor (TNF)-α gene expressions were, respectively, upregulated by ~14-, 22-, 2.2-, 13-, 200-, and 38-fold in miR-122-transfected compared with non-transfected (WT) ZFL cells. In cells transfected with miR-192 and treated with LPS after 8-12?h, IL-22, lysozyme, TLR1, TLR3, TLR4a, and TNF-α expressions significantly differed between WT and miR-192-overexpressing ZFL cells. However, we observed significantly higher IL-22 expression levels after 12?h of LPS treatment in miR-192-transfected ZFL cells compared with non-transfected cells. In contrast, IL-22, lysozyme, and TNF-α were markedly upregulated (>100-fold) after miR-194a transfection and overexpression in ZFL cells and treatment with LPS. Our cloning and expression analyses indicated that miR-122, miR-192, and miR-194a play important roles in zebrafish immunology.