肝脏表达的抗菌肽-2

抗菌肽是在多种生物体中表达的具有抗菌活性的肽类物质的总称.肝脏表达的抗菌肽-2(liver-expressed an-timicrobial peptide-2,LEAP-2)在肝脏中产生后分泌到血液中参与血液循环,从而发挥抗菌活性等作用.本文简要介绍LEAP-2的发现过程,对LEAP-2的基因组成、结构、诱导表达机理、抗菌活性以及基因调控等方面的研究进行综述,并展望LEAP-2可能的生理功能.     

日本鳗鲡TLR3基因的克隆及其免疫功能分析

Toll样受体3(TLR3)是用于识别双链RNA的一种重要模式识别受体。利用RT-PCR和RACE技术克隆了日本鳗鲡TLR3(Aj TLR3)基因c DNA全长序列,并采用实时荧光定量PCR(q RT-PCR)检测分析该基因在日本鳗鲡各组织器官以及体外肝脏细胞的表达水平变化,以期对日本鳗鲡TLR3基因的序列特征及其功能进行研究。结果表明:Aj TLR3基因全长3 383 bp,开放阅读框为2 766 bp,编码921个氨基酸。该蛋白具有16个LRR的胞外结构域、跨膜结构域以及TIR结构域,其中在TIR结构域含有一个高度保守的氨基酸残基Tyr~(778)。Aj TLR3基因在日本鳗鲡各组织器官中均有表达,其中肝脏表达量最高;经poly I∶C刺激后在血、肠、肝脏、脾脏、皮肤、心脏及肌肉组织中均显著提高;而LPS刺激后,仅在肝脏和肠中有显著提高。日本鳗鲡肝脏细胞体外实验结果显示:poly I∶C处理后12 h和24 h表达水平显著增高,Cp G-DNA和肽聚糖处理后24 h表达量均有显著增加;而细菌浓度达到107 CFU/m L和108 CFU/m L后,Aj TLR3基因表达水平分别在24 h、12 h显著增高并达到峰值。以上研究表明,Aj TLR3在日本鳗鲡抵御病毒及细菌的免疫应答过程中具有重要作用。     

青蛤抗菌肽基因的克隆及其在组织间的表达分析

利用构建的SMART-cDNA文库及高通量测序方法,获得了青蛤抗菌肽macin家族相关基因(mytimacin)的全长序列,采用荧光定量PCR方法分析了mytimacin在青蛤各组织的表达情况,并在鳗弧菌胁迫下分析了mytima-cin在外套膜中的时序表达关系。结果表明,mytimacin基因全长461bp,开放阅读框为261bp,编码86个氨基酸,具有24个氨基酸的信号肽序列;荧光定量PCR结果显示,该基因在血液、肝脏、外套膜、鳃和闭壳肌等组织中普遍表达,其中外套膜表达水平最高,在鳃中表达最低;在鳗弧菌刺激后6～24h,青蛤外套膜中mytimacin的表达量出现明显上调的趋势且与对照组差异显著(P<0.05),说明mytimacin抗菌肽基因在青蛤的免疫反应中具有重要作用。     

大黄鱼肝表达抗菌肽2基因的克隆和原核表达

抗菌肽是在多种细胞中表达具有抗菌活性的肽类物质的总称,在免疫反应中发挥着非常重要的作用.通过同源克隆法克隆到大黄鱼肝脏表达的抗菌肽2(liver-expressed antimicrobial peptide-2,LEAP-2)基因的完整开放阅读框(Opening Reading Frame,ORF).克隆到的大黄鱼LEAP-2全长2236 bp,包含外显子Ⅰ78 bp,内含子Ⅰ880 bp,外显子Ⅱ179 bp,内含子Ⅱ1044 bp,外显子Ⅲ55 bp,编码序列312 bp,编码103个氨基酸.推断的氨基酸序列羧基端区域存在高度保守的4 个半胱氨酸残基,符合LEAP-2超家族的结构特征.同源性对比后显示LEAP-2基因在进化上高度保守,大黄鱼LEAP-2推断的氨基酸序列与牙鲆、黄颡鱼、蓝色鲶鱼和斑点叉尾鮰等鱼类之间的同源性均在95%以上.将大黄鱼LEAP-2 cDNA连接到pET-32a(+),构建了重组表达质粒pET-32a-LEAP-2,将其转化到大肠杆菌BL21上并用1.0 mmol/L IPTG诱导表达,获得了大小约为27 kDa的重组蛋白,与预期的一致.     

日本鳗鲡TLR21基因的鉴定、免疫应答与启动子分析

为研究TLR21（Toll like receptor 21）在低等脊椎动物中的功能及表达调控机制,我们扩增获得了日本鳗鲡TLR21（AjTLR21）cDNA序列,其编码的蛋白具有TLR家族的共同特征。AjTLR21基因结构与其他鱼类和两栖类TLR21相同,由单个外显子编码。荧光定量结果显示,AjTLR21在血液、鳃、脾脏、中肾等11个组织/器官中转录表达,其中在血液中表达量最高。经Poly I:C诱导后8h,AjTLR21在脾脏和中肾中的表达量显著性上调;诱导后16h,AjTLR21在血液、鳃、肠和脾脏中的表达量显著性上调（P < 0.05）。双荧光素酶报告基因结果显示,在AjTLR21 5'上游调控序列-1179 bp到+117 bp存在Poly I:C调节的正调控元件。经Edwardsiella tarda诱导后16h和72h,AjTLR21分别在血液和中肾组织的表达量显著性上调,表明AjTLR21同时也参与了抗细菌免疫应答,其在机体免疫系统中的功能具有多样性。研究对于理解日本鳗鲡AjTLR21的免疫学功能具有重要的理论意义和应用价值。     

半滑舌鳎miR-200a和miR-200b前体克隆及其免疫应答分析

为了探究半滑舌鳎（Cynoglossus semilaevis）miR-200a和miR-200b在免疫应答中的作用,采用PCR方法克隆了半滑舌鳎miR-200家族的miR-200a和miR-200b的前体序列,长度分别为82和88 bp;用The mfold Web Server和Clustalx1.83软件对其前体序列进行了二级结构和同源性分析,miR-200a和miR-200b都具有典型的颈环结构,与其他物种具有较高的同源性。qRT-PCR分析结果显示,miR-200a和miR-200b在健康半滑舌鳎13种组织（肝脏、肠、脾脏、头肾、后肾、鳃、血液、脑、皮肤、肌肉、胃、心脏和卵巢）中均有表达,miR-200a在头肾中表达量最高,在血液中表达量最低,miR-200b在肝脏中表达量最高,在肌肉中表达量最低;miR-200a和miR-200b在鳗弧菌（Vibrio anguillarum）感染半滑舌鳎后不同时间点的4种免疫相关组织（肝脏、肠、脾脏和头肾）中的表达呈现出先上调后下降的规律,但表达达到峰值的时间点有所不同。miR-200a在肝脏和脾中的表达峰值出现在鳗弧菌感染后6h,在肠和头肾中则是鳗弧菌感染后12h,miR-200b在肠、脾和头肾中均在鳗弧菌感染后12h达到表达高峰;miR-200a和miR-200b在脂多糖（LPS）、肽聚糖（PGN）、葡聚糖（WGP）、聚肌胞苷酸（poly I:C）4种病原模拟物刺激后的半滑舌鳎肝脏细胞系中呈现出上调表达趋势,其中Poly I:C刺激半滑舌鳎肝脏细胞系后miR-200a上调表达趋势明显,6h的表达量为0h的9倍,在WGP刺激半滑舌鳎肝脏细胞后miR-200b上调表达趋势明显,2h的表达量为0的9倍。研究结果为揭示miRNA在半滑舌鳎免疫应答中的作用提供了科学依据。     

CFTR基因在小鼠肠道组织中的差异表达

目的研究肠道组织CFTR基因表达与分泌性腹泻发生的关系。方法选取KM小鼠24只,雌雄各半,随机分为3组（每组8只）：对照组经小鼠腹腔注射0.2 mL生理盐水,实验组小鼠经腹腔注射LPS[6 mg/（kg·bw）]分别作用1 h、8 h,于注射后通过小鼠精神状态、肠道组织形态学判定分泌性腹泻模型的建立,利用荧光定量PCR法检测各段肠道组织CFTR基因的表达。结果 LPS成功诱导小鼠发生了分泌性腹泻;CFTR基因在小鼠十二指肠、空肠、回肠和结肠组织中均有不同的表达丰度,以结肠最高,但各段肠道间差异不显著;与对照组相比,LPS上调了十二指肠、空肠和回肠CFTR基因的转录,下调了结肠CFTR基因的转录。结论提示肠道组织CFTR基因转录水平的上调与LPS诱导分泌性腹泻的发生密切相关,且在各肠段发挥的作用不同,其中空肠在氯离子（Cl-）分泌中发挥主要作用,结肠的作用最弱。     

日本鳗鲡IFN-γ基因的鉴定、表达模式及启动子活性分析

为揭示鱼类IFN-γ的生物学功能, 研究从日本鳗鲡(Anguilla japonica)中克隆获得了IFN-γ基因, 命名为AjIFN-γ。AjIFN-γ具有脊椎动物IFN-γ的典型特征: 包括4外显子/3内含子的基因结构、C端的IFN-γ特征性氨基酸基序和1个核定位信号, 以及6个α-螺旋反向平行构成的二级结构。AjIFN-γ在日本鳗鲡所有组织中均低水平转录表达, 其中肝脏中表达量最高, 其次是皮肤和头肾。Poly I:C刺激和迟缓爱德华氏菌感染均可显著诱导AjIFN-γ在鳃、头肾、体肾和(或)脾脏中的转录表达, 表明AjIFN-γ能够参与日本鳗鲡抗菌和抗病毒的免疫过程。此外, 研究还克隆了AjIFN-γ基因的5′调控区序列共1536 bp, 并构建了一系列Aj IFN-γ 5′调控区删节突变体, 分析其启动子活性, 结果表明, 上游–240/+136区域中含有起始AjIFN-γ转录的关键启动子调控元件, –1062/–814区域存在转录的正调控元件, 而–1252/–1062区域存在转录的负调控元件。上述结果进一步丰富了鱼类IFN-γ的基础知识。     

青蛤Kazal型丝氨酸蛋白酶抑制剂基因的克隆及其在组织间的表达分析

利用构建的SMART-cDNA文库及高通量测序方法,获得了青蛤Kazal家族丝氨酸蛋白酶抑制剂基因(Serine Protease Inhibitor,SPI)的全长序列,采用荧光定量PCR方法分析了SPI在青蛤各组织的表达情况,并在鳗弧菌胁迫下分析了SPI在青蛤血液中的时序表达关系。结果表明,青蛤SPI基因全长587 bp,CDS为67~520 bp,编码151个氨基酸,具有19个氨基酸的信号肽序列;荧光定量PCR结果显示,该基因在血液、肝脏、外套膜、鳃和闭壳肌等组织中表达差异明显,其中血液中表达水平最高,在闭壳肌中表达水平较低,其他组织表达量极少;在鳗弧菌刺激后3 h和48 h青蛤血液中SPI的表达量出现明显上调的趋势且与对照组有极显著性差异(P0.01),说明SPI基因在青蛤的免疫反应中具有重要的作用。     

LPS下调小鼠肝脏、肠道羧酸酯酶表达

目的:研究细菌脂多糖(LPS)对小鼠肝脏、肠道羧酸酯酶表达及酶活性的影响。方法:小鼠经腹腔注射5.0mg/kg的LPS,对照组给予生理盐水,注射后24h处死小鼠,取肝脏和肠道组织。用qRT-PCR技术检测小鼠肝脏、肠道组织羧酸酯酶1和2(mCES1和mCES2)mRNA水平;Westernblot技术检测小鼠肝脏、肠道组织mCES1和mCES2蛋白表达水平。用分光光度计检测小鼠肝脏、肠道组织羧酸酯酶总活性。结果:LPS显著降低小鼠肝脏、肠道组织羧酸酯酶1和2的mRNA水平(P<0.05),同时LPS也显著降低小鼠肝脏、肠道组织羧酸酯酶的蛋白表达水平及酶活性(P<0.05)。结论:LPS造成的炎症状态可显著降低小鼠肝脏、肠道组织羧酸酯酶的表达及酶活性。     

Binding of Ala-scanning analogs of omega-conotoxin MVIIC to N- and P/Q-type calcium channels

We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP-1 (liver-expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine-rich peptides, a 25-residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP-1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram-positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram-negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose-dependently exhibited sensitivity upon treatment with synthetic LEAP-1. The discovery of LEAP-1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.     

Cloning and characterization of the IkappaBalpha gene from Japanese flounder, Paralichthys olivaceus

We identified and characterized the Japanese flounder (Paralichthys olivaceus) inhibitor kappa B alpha (JFIKBA) cDNA. The JFIKBA cDNA contains an open reading frame of 960bp encoding 320 amino acid residues. JFIKBA contains 6 ankyrin repeats in the central coding region. Expression studies by RT-PCR showed constitutive expression of the JFIKBA gene in several Japanese flounder tissues (brain, muscle, gill, heart, kidney, liver, spleen and intestine). Moreover, expression of JFIKBA mRNA was induced in kidney by LPS stimulation. To investigate the role of JFIKBA, we constructed a recombinant plasmid expressing the JFIKBA coding region under the control of the cytomegalovirus (CMV) promoter. Over-expression of the JFIKBA gene in the Japanese flounder cultured cell line derived from kidney, suppressed the expression of the TNF alpha gene with lipopolysaccharide stimulation. These results indicated that JFIKBA has an important role in the innate immune system, especially in the signaling of the cytokine network.     

BIRC7 gene in channel catfish (Ictalurus punctatus): identification and expression analysis in response to Edwardsiella tarda, Streptococcus iniae and Channel catfish Hemorrhage Reovirus

A family member of inhibitor of apoptosis protein (IAP) termed baculoviral IAP repeat-containing 7 (BIRC7) from channel catfish (Ictalurus punctatus) was identified, the full length cDNA sequence of channel catfish BIRC7 (CcBIRC7) was 1686?bp, containing a 5'UTR of 93?bp, a 3'UTR of 399?bp with a poly (A) tail and an ORF of 1194?bp encoding a putative protein of 398 amino acids. The putative CcBIRC7 protein contains two BIR super-family conservative domains and a C-terminal RING finger motif. Phylogenetic analysis showed that catfish CcBIRC7 was moderately conserved with other BIRC7. Quantitative real-time PCR was conducted to examine the expression profiles of CcBIRC7 in healthy tissues and responding to different pathogens (Edwardsiella tarda, Streptococcus iniae and Channel catfish Hemorrhage Reovirus (CCRV)). CcBIRC7 was widely expressed in healthy tissues of channel catfish and with the highest 37.28-fold expression in blood. E.?tarda and S.?iniae could induce CcBIRC7 gene expression drastically in head kidney, liver and spleen, which the peak value reached 31.6-fold, 613.9-fold and 34.4-fold increase by E.?tarda infection, and 248.3-fold, 1540.3-fold and 120.4-fold increase post S.?iniae challenge, respectively. While, CCRV virus could slightly induce CcBIRC7 expression in head kidney and liver but reduce it in spleen. The result suggested BIRC7 may play a potential role in channel catfish innate immune system against bacterial and virus infections, especially as the anti-bacteria immune gene. This is the first report of BIRC7 gene identification and its expression in fish.     

Identification of two insulin-like growth factor IIs in the Japanese eel, Anguilla japonica: cloning, tissue distribution, and expression after growth hormone treatment and seawater acclimation

To better understand the role of IGFs in Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-II (IGF-II) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-II cDNAs, eIGF-II-1 and eIGF-II-2, were cloned from the liver. A signal peptide and a mature peptide of both preproIGF-IIs were composed of 47 amino acids (aa) and 69 aa, but they differed at 17 aa and 13 aa, respectively. The E domain of eIGF-II-1 was 49 aa longer than that of eIGF-II-2, and differed at 22 aa within 52 aa. The highest eIGF-II-1 and II-2 mRNA levels were observed in the liver, with detectable levels also found in all tissues examined. The eIGF-II-1 mRNA levels in the liver, heart, and muscle were higher in females than in males, whereas those in the stomach and intestine were lower in the females. The eIGF-II-2 mRNA levels in the liver and swim-bladder were also higher in females than in males whereas those in the stomach, spleen, and intestine were lower in the females. The eIGF-II-1 mRNA levels in the liver were higher in large compared to small glass eels, while the eIGF-II-2 mRNA levels did not correlate with body weight. Both eIGF-II mRNA levels in the liver increased after eel GH treatments in vivo and in vitro. No differences in both eIGF-II mRNA levels were observed in the gills, liver, stomach and whole kidney between seawater- and freshwater-reared eels.