The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

The effects of CpG-C oligodeoxynucleotides on innate immune responses in Eriocheir sinensis (H. Milne-Edwards, 1853)

CpG oligodeoxynucleotides (ODNs) can stimulate the immune system, and therefore are widely used as a therapeutic vaccination and immune adjuvant in human. In the present study, CpG-C, a combination of A- and B-class ODN, was injected into Chinese mitten crab Eriocheir sinensis at three doses (0.1, 1 and 10 mu g crab-1), and the reactive oxygen species (ROS) levels, activities of total intracellular phenoloxidase (PO) and lysozyme-like activities, the mRNA transcripts of EsproPO, EsCrustin and EsALF were assayed to evaluate its modulating effects on the immune system of crab. The ROS levels in all treated and control groups were significantly increased from 6 to 24 h, except that ROS in 0.1 mu g CpG-C-treated crabs was comparable to that of the blank at 6 h. The PO activity was significantly enhanced and EsproPO transcripts were down-regulated (P < 0.01) at 6 h after the injection of 0.1 mu g CpG-C, with no significant changes in the other dosage treatments. The lysozyme-like activities and EsCrustin transcripts in the CpG-C-treatment groups were significantly higher than those of controls. The mRNA expression of EsALF remained almost constant in all the groups during the treatment. These results collectively suggested that CpG-C could activate the immune responses of E. sinensis, and might be used as a novel immunostimulant for disease control in crabs.

Molecular cloning and characterization of peroxiredoxin 6 from Chinese mitten crab Eriocheir sinensis

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). In this study, the full-length cDNA encoding peroxiredoxin 6 (designated EsPrx6) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5' untranslated region (UTR) of 69 bp, a 3' UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. Quantitative real-time RT-PCR was employed to assess the mRNA expression of EsPrx6 in various tissues and its temporal expression in haemocytes of crabs challenged with Listonella anguillarum. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12 h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146 bp with a 5' UTR of 103 bp, an unusually long 31 UTR of 1519 bp with a canonical polyadenylation signal sequence AATAAA and a potyA tail, and an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5 kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12 h post-Vibrio infection, then recovered to the original level at 24 h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop. (c) 2007 Published by Elsevier Ltd.

The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri

Cu, Zn superoxide dismutases (SODs) are rnetalloenzymes that represent one important line of defence against reactive oxygen species (ROS). A cytoplasmic Cu. Zn SOD cDNA sequence was cloned from scallop Chlamys farreri by the homology-based cloning technique. The full-length cDNA of scallop cytoplasmic Cu, Zn SOD (designated CfSOD) was 1022 bp with a 459 bp open reading frame encoding a polypeptide of 153 amino acids. The predicted amino acid sequence of CfSOD shared high identity with cytoplasmic Cu. Zn SOD in molluscs, insects, mammals and other animals, such as cytoplasmic Cu, Zn SOD in oyster Crassostrea sostrea gigas (CAD42722), mosquito Aedes aegypti (ABF18094), and cow Bos taurus (XP_584414). A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with Listonella anguillarum, Micrococcus luteus and Candida lipolytica respectively. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD dropped in the first 8-16 h and then recovered after challenge with L. anguillarum and M. litteus, but no change was induced by the C. lipolytica challenge. The results indicated that CfSOD was a constitutive and inducible acute-phase protein, and could play an important role in the immune responses against L. anguillarum and M. luteus infection. (C) 2007 Elsevier Ltd. All rights reserved.

中华绒螯蟹（Eriocheir sinensis）Peroxiredoxin 6和Thioredoxin1基因的克隆及表达

中华绒螯蟹是我国重要的水产经济动物，近年来养殖规模不断扩大，产量持续增加。但是，伴随着养殖规模的扩大，养殖环境也日益恶化并导致了大量疾病的发生，严重制约了中华绒螯蟹养殖业的健康发展。因此，疾病预防和控制对中华绒螯蟹养殖业的可持续发展具有举足轻重的作用。与其他无脊椎动物一样，中华绒螯蟹的免疫系统没有免疫球蛋白和淋巴细胞，而是依靠由细胞免疫和体液免疫构成的固有免疫系统来对病原进行识别和清除。中华绒螯蟹的固有免疫机制的研究有助于推动中华绒螯蟹病害防治工作的开展。 本研究采用大规模EST测序方法，结合末端快速扩增（rapid amplification of cDNA ends，RACE）技术从中华绒螯蟹血淋巴中克隆到了过氧化物还原酶（peroxiredoxin，EsPrx6）和硫氧还蛋白（thioredoxin，EsTrx1）基因的cDNA 全长序列；采用实时荧光定量PCR 技术检测了这两个基因在健康个体中表达的组织分布情况以及鳗弧菌刺激后血淋巴细胞中的时序表达规律；同时，将这两个基因的编码区克隆到pET 系列载体，并在大肠杆菌中实现了重组表达，并进行了体外活性检测。 过氧化物还原酶是一个抗氧化蛋白超家族，在保护机体免受活性氧（reactive oxygen species，ROS）的伤害中发挥着重要作用。中华绒螯蟹Prx6（EsPrx6） 基因的cDNA 全长为1076 bp，5` UTR（untranslated region，UTR） 为69 bp，3` UTR 为347 bp，开放阅读框（open reading frame，ORF）为660 bp，编码219 个氨基酸的蛋白。mRNA 3`-端具有多聚腺苷酸加尾信号（polyadenylation signal）AATAAA 和polyA 尾巴。EsPrx6 的预测分子量为 24 kDa，理论等电点为6.21，具有一个保守的Prx 结构域、一个AhpC 结构域和过氧化物酶催化活性中心PVCTTE，表明EsPrx6 属于1-Cys 型Prx。在所检测的组织中均有EsPrx6 的表达，其中以肝胰腺表达量最高，为血淋巴细胞中表达量的17.4 倍。鳗弧菌刺激后，血淋巴细胞中EsPrx6 的表达下降，到12 h 时，实验组显著低于对照组（P<0.05）；随时间推移，表达水平逐渐回升，但在整个实验期间，都没有恢复到起始水平。将EsPrx6 进行体外重组并在大肠杆菌E. coli BL21（DE3）中实现表达，重组EsPrx6 具有预期的抗氧化活性和过氧化物酶活性，其中抗氧化活力为14.69 U/mg 蛋白，高于相同条件下GSH 的抗氧化力（P<0.05），过氧化物酶活力为23.46 U/mg 蛋白。结果表明，EsPrx6 作为一种重要的抗氧化剂，在中华绒螯蟹抵御ROS 可能引起的氧化损伤方面具有重要作用。 硫氧还蛋白是广泛存在于生物体内的一种具有硫醇依赖性的具有还原活性的蛋白。中华绒螯蟹Trx1（EsTrx1）基因的cDNA 全长为641 bp，5` UTR 为17 bp，3` UTR 为306 bp，开放阅读框为318 bp，编码105 个氨基酸。EsTrx1 的预测分子量为12.2 kDa，理论等电点为4.8。EsTrx1 不含信号肽，其氨基酸序列与其他动物的Trx1s 具有高度相似性，如与地中海黄蝎的Trx1 相似度达到73%；而与其他物种Trx2 的同源性很低，相似度仅为14.3-22.8%，表明EsTrx1 属于Trx1 亚族。实时荧光定量PCR 检测发现，EsTrx1 在鳃、性腺、肝胰腺、肌肉、心脏和血淋巴细胞中都有表达。血淋巴细胞中EsTrx1 mRNA 的表达量在菌刺激后上升，刺激后6 h，实验组表达量显著高于对照组和空白组（P<0.05），然后逐渐恢复到刺激前水平。为进一步探讨其生物学功能，将EsTrx1 进行体外重组并在大肠杆菌E. coli BL21（DE3）得到表达，重组EsTrx1 具有预期的氧化还原调节活性，抗氧化活力为3.06 U/mg，且抗氧化活力高于GSH（P<0.05）。rEsTrx1 的二硫键还原活力为5.03，低于凡纳滨对虾的二硫键还原活力（10.44），接近于大肠杆菌（4.93），小牛胸腺（6.50）和小牛肝脏（5.09），而高于鲍鱼Trx2（1.83）活力。结果表明，EsTrx1 在生理条件下能够作为一种重要的抗氧化剂，参与对细菌感染的免疫应答反应。

中国明对虾免疫系统中抗氧化相关基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明，当对虾等甲壳动物受到外界病原刺激时，其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢，引起呼吸爆发，产生多种活性氧分子。另外，受到病原侵染的对虾还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物，但同时由于活性氧分子反应的非特异性，它们也会对宿主的细胞、组织和器官造成严重伤害，进而导致对虾生理机能的损伤和免疫系统的破坏。所以，消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力，提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶（mMnSOD）、胞质型超氧化物歧化酶（cMnSOD）、过氧化氢酶（Catalase）和过氧化物还原酶（Peroxiredoxin）等四种与免疫系统相关的抗氧化酶基因，分析了它们的分子结构特征，组织分布及应答不同病原刺激的表达变化模式，并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶（SOD）基因，通过序列比对分析发现，其中一个为mMnSOD基因，另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基，其中开放阅读框为660个碱基，编码220个氨基酸，其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明，该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示，对虾感染病毒3 h时，该基因在血细胞和肝胰脏中的转录水平显著升高。此外，通过构建原核表达载体，本研究对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基，其中开放阅读框为861个碱基，编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示，cMnSOD基因在对虾被检测的各个组织中均有表达。 另外，半定量RT-PCR结果表明，对虾感染病毒23h时，该基因在肝胰脏中的转录上升到正常水平的3.5倍；而感染后59 h时，该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物，结合RACE技术，从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段，片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现，该基因在肝胰脏、鳃、肠和血细胞中表达水平较高，在卵巢、淋巴器官和肌肉中的表达水平相对较弱；感染病毒23 h和37 h时，对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息，结合SMART-RACE技术，从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因（Peroxiredoxin）， 该基因的cDNA全长为942个碱基，其中开放阅读框为594个碱基，编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da，pI为5.17。Northern blot结果表明，Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示，弧菌感染后，该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外，对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明，复性后的重组蛋白能在DTT存在的条件下还原H2O2。

皱纹盘鲍血细胞内活性氧抗病机制及几种环境污染物及其影响的研究

本研究首次揭示了皱纹盘鲍(Haliotis discus hannai) 血细胞内存在着利用活性氧（Reactive oxygen species, ROS）的抗病机制。为今后我国研究贝类的活性氧抗病机制和筛选免疫药物提供了理论基础。本研究主要结果如下：1. 利用鲁米诺（Luminol, 3-氨基邻苯二甲酰肼）为依赖的化学发光法在体外条件下用不同刺激物（酵母细胞和酵母聚糖）对皱纹盘鲍血细胞进行刺激，测定血细胞吞噬活动中历经呼吸爆发产生活性氧的化学发光反应。结果表明皱纹盘鲍血细胞在体外条件下，经刺激物诱导吞噬活动中有明显的呼吸爆现象和很强的活性氧产生。不同有刺激物诱导血细胞产生的化学发光强度不同；同一刺激物的不同处理和不同浓度对血细胞产生活性氧的化学发光强度的影响不同。刺激物经皱纹盘鲍自体血清调理和未经调理对血细胞刺激所产生的化学发光强度不同。SOD和NaN_3对皱纹盘鲍血细胞吞噬过程中活性氧产生的化学发光有抑制作用。上述结果证明了皱纹盘鲍血细胞内存在有象高等动物血细胞内一样的MPO-H_2O-卤素系统的氧化性抗病机制，即在血细胞吞噬异物过程中能够释放活性氧（ROS）对异物进行杀灭的功能。2. 利用活性氧清除剂（过氧化氢酶、SOD、苯甲酸钠、2，5-二甲基呋喃、NBT和EDTA等）对皱纹盘鲍血细胞释放的活性氧进行分类测试，结果表明活性氧清除剂对皱纹盘鲍血细胞吞噬的化学发光都有明显的抑制作用，从而证明皱纹盘鲍血细胞能够释放的活性氧的种类有：超氧阴离子（O_2~-），过氧化氢（H_2O_2），羟自由基（OH~·）和单线态氧（~1O_2）。3. 在体外条件下利用化学发光法定量地研究了不同种类和不同浓度的农药：对硫磷（Parathion）、敌敌畏（Dichlorovos)、乐果（Dimethoate)、2，4-D 丁酯(2,4-D butylester)和甲胺磷（Methamidophos）对皱纹盘鲍血细胞氧化性抗病机制的影响。结果显示不同农药对皱纹鲍血细胞产生ROS的影响程度不同，及同一种农药的不同浓度的浸泡1h或浸泡12h处理皱纹盘鲍血细胞都能够抑制血细胞吞噬时的化学发光，表明农药能够抑制皱纹敌国鲍血细胞吞噬活动中的活性氧（ROS）的产生，而且这种抑制作用随着农药浓度的增加而加强。几种农药抑制皱纹盘鲍血细胞产生活性氧（ROS）的强度不同：2，4D-丁酯，敌敌畏和乐果的抑制作用强于对硫磷和甲胺磷。同时测定了不同浓度的农药浸泡12h后的皱纹盘鲍因细胞吞噬酵母细胞的吞噬百分率和吞噬指数，结果显示多数农药在低浓度时能够提高皱纹盘鲍血细胞的吞噬百分率和吞噬指数，而在高浓度时则能抑制血细胞吞噬酵母细胞的活力而降低血细胞的吞噬百分率的吞噬指数。4. 在体外条件下利用化学发光法定量地研究了不同种类和不同浓度的重金属：铭（Cr）、镉（Cd）、汞（Hg）、铅（Pb）、铜（Cu）和锌（Zn）对皱纹盘鲍血细胞氧化性抗病机制的影响。结果显示不同种类和不同浓度的重金属浸泡1hr.处理皱纹盘鲍血细胞都不同程度地抑制了血细胞吞噬时的化学发光，表明重金属能够抑制皱纹盘鲍血细胞吞噬活动中的活性氧（ROS）的产生，而且这种抑制作用随着重金属浓度的增加而加强。不同的重金属抑制强度不同，从强到弱依次为Hg > Cd > Cu > Pb > Cr > Zn. 研究表明六种重金属中，Hg对皱纹盘鲍血细胞的免疫毒性最大。

栉孔扇贝和海湾扇贝免疫防御特性的比较研究

贝类养殖作为我国传统的海水养殖产业，在我国沿海地区的经济发展中起重要作用。但由于近年来养殖扇贝病害的不断发生，不仅造成了巨大的经济损失，而且直接威胁到现有养殖产业的生存和发展。目前认为，机体抗病能力的下降是发生流行性死亡的主要原因之一。因此，深入研究扇贝免疫防御特性，能更好地了解和解决贝类养殖中的病害问题。本文比较了健康状态和重金属污染以及高温胁迫下海湾扇贝和栉孔扇贝部分免疫指标的变化，并对同龄不同大小的栉孔扇贝的部分免疫指标进行了比较，以期更好的了解贝类的防御机制，为扇贝病害防治提供资料。研究结果如下: 用流式细胞仪技术对血细胞死亡率、细胞吞噬率和呼吸爆发进行了测定，发现健康状态下两种扇贝的细胞死亡率相差不大，海湾扇贝为4.83%，栉孔扇贝为4.95%；细胞吞噬率差异显著(P<0.05)，海湾扇贝为26.73%，栉孔扇贝为19.89%；呼吸爆发的基础值相差也不大，海湾扇贝为1.94，栉孔扇贝为1.56。另外用免疫化学方法对健康状态下栉孔扇贝和海湾扇贝血淋巴和肝胰腺中SOD、ACP活性和MDA含量也进行了测定与比较。发现在血淋巴和肝胰腺中，两种扇贝SOD活性均存在显著差异(P<0.05)，其中海湾扇贝分别为995.43和113.99，栉孔扇贝分别为446.64和40.6；血淋巴和肝胰腺中ACP活性差异显著(P<0.05)，海湾扇贝分别为90.19和432.36，栉孔扇贝分别为73.26和146.63；血淋巴中和肝胰腺中MDA含量差异不显著(P>0.05)，海湾扇贝分别为3.37和92.46，栉孔扇贝分别为2.17和28.96。实验结果说明，海湾扇贝对细菌等异物的吞噬和杀灭能力以及机体自身的抗氧化能力高于栉孔扇贝，这为海湾扇贝比栉孔扇贝具有更高的抗逆性提供了证据。 用相同的方法对不同浓度(0, 0.2, 0.3, 0.5 mg L-1) Pb2+溶液浸泡后的栉孔扇贝和海湾扇贝的血细胞死亡率、细胞吞噬率和呼吸爆发进行了检测，同时测定了Pb2+处理后肝胰腺中SOD、ACP活性和MDA含量的变化。结果表明，栉孔扇贝的血细胞死亡率与对照组相比有上升趋势，且随着Pb2+浓度的增加而显著增加，而海湾扇贝在相同条件Pb2+的胁迫下，血细胞的死亡率明显低于栉孔扇贝。栉孔扇贝各实验组细胞吞噬率均低于对照组，而海湾扇贝在低浓度的Pb2+浸泡后，细胞的吞噬率与对照组相比有所降低，但随着浓度的继续增加，细胞的吞噬率反而上升，甚至略高于对照组。栉孔扇贝血细胞的呼吸爆发在Pb2+胁迫后较对照组有所升高，但随着浓度的增加，呼吸爆发有下降的趋势，而海湾扇贝的呼吸爆发随着Pb2+浓度的增加而显著增加。对两种扇贝体液免疫指标的测定发现，各实验组SOD的活性均比对照组高，且海湾扇贝的SOD活性高于栉孔扇贝；随着Pb2+浓度的增加，各实验组ACP的活性一直呈上升趋势，且海湾扇贝的ACP活性也高于栉孔扇贝；Pb2+处理后MDA的含量均显著增加，其中栉孔扇贝MDA含量的增幅明显高于海湾扇贝。该结果说明，Pb2+对两种扇贝的免疫系统都有一定的影响，但相同剂量的Pb2+对两种扇贝的毒害程度不同，海湾扇贝对Pb2+的耐受性似乎高于栉孔扇贝。 对海湾扇贝和栉孔扇贝的升温刺激实验发现，当养殖水体的温度从18℃缓慢升高到30℃后，栉孔扇贝在第三天开始出现大量死亡现象，而海湾扇贝未出现死亡。因而本文只对升温前后的海湾扇贝进行了细胞死亡率、吞噬率和呼吸爆发的测定和比较。结果发现，与升温前相比，血细胞的死亡率在温度升高后略有增加，但差异不显著(P>0.05)；而细胞吞噬率在温度升高后显著下降；通过对呼吸爆发积聚的活性氧（Reactive Oxygen Species，ROS）的测定发现，升温后血细胞的ROS含量显著高于升温前(P<0.05)。该结果表明，高温会影响血细胞的功能，使机体的免疫防御能力下降，从而导致病害的发生。 另外，本文对同龄不同大小的栉孔扇贝的血细胞吞噬率以及血淋巴和肝胰腺中SOD、ACP活性和MDA含量进行了测定与比较。结果发现: 小扇贝的血细胞吞噬率略高于大扇贝，分别达到了22.82和20.96；在血淋巴及肝胰脏中，两组扇贝SOD酶的活性均差异显著(P<0.05)，小扇贝分别为613.88和102.01，大扇贝分别为356.15和50.22；血淋巴中小扇贝ACP的活性极显著的高于大扇贝(P<0.01)，分别为44.3和2.91，但肝胰腺中大扇贝和小扇贝ACP的活性相差不大，大扇贝为455.75，小扇贝为485.33；大扇贝和小扇贝血淋巴和肝胰脏中MDA含量相差不大，大扇贝分别为2.67和31.83，小扇贝分别为3.17和35.33。该结果说明，小扇贝对细菌等异物的吞噬和杀灭能力以及机体自身的抗氧化能力高于大扇贝，这为生长与抗逆性相颉抗提供了依据。

海湾扇贝超氧化物歧化酶家族基因结构、表达和多态性分析

海湾扇贝Argopecten irradian Lamarck于1982年从美国引种到中国，由于具有较快的生长速度和很高的经济效益，海湾扇贝成为中国最主要的养殖贝类之一。近年来海湾扇贝养殖遇到了死亡率高等问题，深入开展海湾扇贝功能基因的研究，尤其是免疫相关基因及其机制研究并在此基础上寻找扇贝疾病防治的有效方法对海湾扇贝的健康养殖十分重要。 对于贝类免疫系统来说，其血细胞在先天性免疫防御中起着重要的作用。当受到外界病原侵染时，贝类血细胞的一个重要免疫反应就是吞噬作用。在吞噬病原过程中，受到病原侵染的贝类还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发与呼吸链相耦联的活性氧（ROS）的大量产生。这些活性氧具有极强的反应特性，能破坏病原微生物的结构和功能分子，实现对入侵病原的杀灭。利用活性氧对被吞噬的病原进行杀灭，这是吞噬作用消除病原抵御侵染的重要机制。但由于活性氧分子反应的非特异性，它们也会破坏宿主机体细胞内的功能蛋白分子、不饱和脂肪酸分子和核酸等，对细胞造成严重的伤害，进而导致机体生理机能的损伤和免疫系统的破坏。所以，及时消除病原感染机体内过量产生的ROS，维持相关细胞的正常代谢，对提高机体抵抗力和免疫力具有重要的作用。O2-是生物体内产生的第一种活性氧分子，其他的活性氧分子也是由它衍生而来，消除过量O2-是消除过量活性氧危害的第一步也是关键一步。生物体内，超氧化物歧化酶（SOD）是催化O2-发生歧化反应，消除O2-的关键酶。 首先，本文通过RACE方法获得了海湾扇贝SOD家族全部三种基因的cDNA全长并对其进行了序列的生物信息学分析，海湾扇贝AiCuZnSOD全长cDNA为1047个碱基，其中开放阅读框为459个碱基，编码152个氨基酸，与栉孔扇贝Chlamys farreri的CuZnSOD相似度为77.5%，与长牡蛎Crassostrea gigas的相似度为75%，与人的相似度为74.7%。AiMnSOD全长cDNA为1207个碱基，其中开放阅读框为678个碱基，编码226个氨基酸，序列比对结果发现AiMnSOD的氨基酸序列与虾夷扇贝Mizuhopecten yessoensis和皱纹盘鲍Haliotis discus hannai的相似度分别为85%和78.4%，与哺乳动物相似度也在68%~72%之间。AiECSOD全长cDNA为893个碱基，其中开放阅读框为657个碱基，编码218个氨基酸。AiECSOD与其它物种ECSOD相似度比较低。与线虫Brugia pahangi的相似度为27.9%，与疟蚊Anopheles gambiae的相似度为31.4%，与斑马鱼Danio rerio的相似度为27.8%，与人的相似度也只有28.6%，与同是贝类的长牡蛎ECSOD也只有28.1%的相似性。主要原因是AiECSOD的信号肽和肝磷脂结合区域在各物种中无同源性。 其次，采用qRT-PCR（quantitative real time PCR）方法分析三种SOD基因在不同组织中的表达情况，结果表明三种SOD基因的组织表达有所差异。AiCuZnSOD基因在鳃中表达水平最高，其次是血细胞和性腺，在外套膜、闭壳肌和肝胰脏表达水平较低。AiMnSOD基因在鳃中表达水平最高，其次是外套膜，在血细胞、性腺，而在肝胰脏和闭壳肌表达较弱。AiECSOD基因在血细胞中表达水平最高，其次是肝胰脏，在鳃、闭壳肌表达水平较低，而性腺和外套膜没有检测到。同时，采用qRT-PCR对鳗弧菌Vibrio angullarum感染后海湾扇贝血细胞中三种SOD基因mRNA表达变化进行了检测。AiCuZnSOD表达量在各个时间段没有显著差异(P > 0.05)。AiMnSOD的表达量在1.5 h时略有下降，在3 h时达到最高表达量，是空白组（0h）的3倍(P < 0.01)，从6 h到24 h表达量逐渐下降，24 h时表达量是空白组的1.6倍，24 h到48 h又稍有升高。AiECSOD的表达量在1.5 h时有所下降，是空白组的0.3倍（P < 0.05)，随后逐渐升高，在12 h时达到最高表达量，是空白组（0h）的4.5倍（P < 0.01），从24 h到48 h表达量逐渐下降并恢复到空白组的水平。在对照组，各个时间点没有显著差异（P > 0.05）。在鳗弧菌感染后，海湾扇贝三种SOD的表达并不一致，且差异比较显著。AiCuZnSOD被认为是构成性表达基因，其受外界刺激的影响最小，AiMnSOD和AiECSOD受刺激后表达上调比较明显。 第三，采用Genome-walking的方法得到了海湾扇贝三种SOD基因的基因组全长和近端启动子序列并对其进行了相关分析。AiCuZnSOD的基因组序列全长为4279bp，包含有4个外显子和3个内含子。AiMnSOD的基因组序列全长为10692bp，包含有4个外显子和3个内含子。AiECSOD的基因组序列全长为5276bp，包含有5个外显子和4个内含子。三种基因外显子和内含子的结合处序列遵循-AT/GT-原则。我们把海湾扇贝SOD家族的三个基因的近端启动子进行了比较分析。发现三种SOD在靠近起始密码子的位置都有Oct-1结合位点。三种SOD共有的转录位点有：Oct-1、C/EBPalp、Oct2.1、Sp-1和GATA-1。AiCuZnSOD和AiMnSOD共有的转录位点有：ICSBP、Ftz、TATA-box、C/EBPbeta和Antp。AiCuZnSOD和AiECSOD共有的转录位点有：AP-1和NFκB。AiMnSOD和AiECSOD共有的转录位点有：GR和ER。AiCuZnSOD独有的位点有：SRF、YY-1和NF-1。AiMnSOD独有的位点有：HNF-1、Hb、MEB、NF-muE1、Pit-1a和Eve。AiECSOD独有的位点有：CREB、RATA-alph、Kruppel-like和AP-3。 此外，通过构建原核表达载体，本研究对AiCuZnSOD和AiECSOD基因进行了体外重组表达，并对纯化的重组蛋白进行了酶活分析。酶活分析表明，重组AiCuZnSOD蛋白有较高的酶活和稳定性。 最后，我们对海湾扇贝三种SOD基因的部分区域，包括启动子、编码区，部分内含子区域进行了SNP检测，并对SOD基因部分SNP位点多态性和鳗弧菌敏感性进行了相关分析。三种SOD基因中，我们共发现了59个SNP位点，其中AiECSOD的SNP位点最多，特别是在启动子区，AiCuZnSOD和AiMnSOD多态性较低。其中AiCuZnSOD启动子区的-1739 T-C 位点的基因型和等位基因，AiECSOD启动子区的-498 A-T和-267 G-A等位基因频率，AiECSOD的第一个外显子38 Thr-Lys的多态性在敏感和抗菌群体中存在显著差异（P < 0.05）。

中国对虾血细胞体外吞噬活动中活性氧产生的研究

该文首先对中国对虾血细胞体外短时培养条件进行了摸索,以期为后来在体外研究中国对虾血细胞吞噬活动中ROS的产生建立基础.在上在实验的基础上,该文利用化学发光法对中国对虾血细胞体外吞噬过程中ROS的产生进行了研究,试图了解血细胞吞噬活动中的化学发光现象.最后利用NBT还原法研究了中国对虾血细胞体外吞噬过程中O<'-><,2>的产生化及一些环境污染物如重金属离子和农药对其吞噬活动中O<'-><,2>的产生影响.

Immune condition of Chlamys farreri in response to acute temperature challenge

The effects of acute temperature challenge on some immune parameters of haemocyte in Zhikong scallop, Chlamys farreri, recognised as a temperature sensitive bivalve species, were evaluated over a short period of time. Scallops were suddenly transferred from 17 degrees C to 11 degrees C, 23 degrees C and 28 degrees C for a period of 72 h. Total haemocyte count (THC), percentage of phagocytic haemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of temperature stress. Results demonstrated that the percentage of phagocytic haemocytes and ACP activity in cell-free haemolymph of scallops challenged at 28 degrees C for 72 h significantly decreased. By contrast, reactive oxygen species production by haemocytes increased when compared to the initial values. It is concluded that haemocyte activities of C. farreri appear to be compromised when scallops were transferred from 17 degrees C to 28 degrees C. Meanwhile, no obvious negative effect of acute temperature stress was detected on haemocyte activities of C. farreri challenged at 11 degrees C, which highlighted the high tolerance of scallops to acute decrease of seawater temperatures. (C) 2007 Elsevier B.V. All rights reserved.

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 degrees C, 17 degrees C and 25 degrees C respectively. Thereafter, a recovery period of 24 h at 17 degrees C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immumomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 degrees C and 24 h of air exposure at 17 degrees C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 degrees C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 degrees C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 degrees C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops. (c) 2007 Elsevier B.V. All rights reserved.

Effect of alginic acid decomposing bacterium on the growth of Laminaria japonica (Phaeophyceae)

We collected the diseased blades of Laminaria japonica from Yantai Sea Farm from October to December 2002, and the alginic acid decomposing bacterium on the diseased blade was isolated and purified, and was identified as Alterornonas espejiana. This bacterium was applied as the causative pathogen to infect the blades of L. japonica under laboratory conditions. The aim of the present study was to identify the effects of the bacterium on the growth of L. japonica, and to find the possibly effective mechanism. Results showed that: (1) The blades of L. japonica exhibited symptoms of lesion, bleaching and deterioration when infected by the bacterium, and their growth and photosynthesis were dramatically suppressed. At the same time, the reactive oxygen species (ROS) generation enhanced obviously, and the relative membrane permeability increased significantly. The contents of malonaldehyde (MDA) and free fatty acid in the microsomol membrane greatly elevated, but the phospholipid content decreased. Result suggested an obvious peroxidation and deesterrification in the blades of L. japonica when infected by the bacterium. (2) The simultaneous assay on the antioxidant enzyme activities demonstrated that superoxide dismutase (SOD) and catalase (CAT) increased greatly when infected by the bacterium, but glutathione peroxidase (Gpx) and ascorbate peroxidase (APX) did not exhibit active responses to the bacterium throughout the experiment. (3) The histomorphological observations gave a distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population on the blades after infection. The bacterium firstly invaded into the endodermis of L. japonica and gathered around there, and then resulted in the membrane damage, cells corruption and ultimately, the death of L. japonica.

STARVATION-INDUCED CHANGES OF HEMOCYTE PARAMETERS IN THE ZHIKONG SCALLOP CHLAMYS FARRERI

Substantial nutritional and energetic demands m-e associated with immune activation and the maintenance of an efficient immune system. One-year-old Chlamys farreri (Jones and Preston) scallops were maintained ill lantern nets ill different nutritional conditions (satiation and starvation) for 40 days. After the 40-day treatments, the condition index and the total hemocyte count (THC) decreased significantly in the starved group compared with the satiated and initial control groups. The percentage of phagocytic hemocytes also was significantly reduced with starvation. In contrast. no significant effect of starvation was observed oil reactive oxygen species (ROS) production. The acid phosphatase (ACP) activities in cell-free hemolymph increased significantly in scallops in starved and satiated treatments compared with the initial control. whereas ACP activity in hemocyte lysate was significantly lower ill the starved group. These results indicate that starvation stress compromises immunological activities of scallops.