鳗弧菌rpoS功能的研究

rpoS 基因编码RpoS因子（RNA聚合酶的σ亚基），能增强细菌细胞对外界不良环境的抗逆性和适应能力。我们从鳗弧菌（Vibrio anguillarum）M3 Fosmid 文库中得到rpoS 基因序列，全长999bp，同源分析发现与大肠杆菌（Escherichia coli）有71%的相似性，与霍乱弧菌（Vibrio cholerae ）有78%的相似性。为了研究rpoS在鳗弧菌中的作用，我们利用in-frame deletion技术构建了rpoS 基因的非极性缺失突变株，同时利用细菌的接合转移，以低拷贝克隆质粒pSUP202为载体，构建了突变株的互补株。 在TSB培养基中，rpoS 基因的缺失减缓了鳗弧菌对数期的生长，但是对稳定期的生长没有影响。对数期鳗弧菌在1M蔗糖（渗透压胁迫）和5%（v/v）乙醇中的生长减缓，而在18%(v/v)乙醇中的生长及42℃热击时的存活率相对于野生型没有变化。不同生长时期的鳗弧菌对15mM H2O2的反应有所不同，rpoS 的缺失使对数期的鳗弧菌对15mM H2O2的反应更加敏感。在rpoS基因互补株中，上述表型几乎恢复到野生型水平。 我们通过感染实验发现，rpoS 基因的缺失使鳗弧菌的LD50提高了20倍。 RpoS的突变对鳗弧菌的泳动和胞外酶的产生也有影响。突变株泳动圈直径是野生型的73.8%，在明胶平板和酪蛋白平板上的透明圈直径分别为野生型的61%和69%。通过azocaseion检测胞外产物ECP酶活发现，突变株的酶活是野生型的35.5%。 以上数据表明了RpoS在鳗弧菌对数期应对外界不良环境时起到了重要作用，同时参与了鳗弧菌的致病过程。

大型藻类在工程治理海水富营养化和抑制病原微生物中的作用

沿海工农业生产的快速发展和人类活动对近海生态系统产生了很大影响，大量化肥的使用和工业污水、生活污水的排放导致近海环境污染，海水富营养化，赤潮频发。另外，由于近海养殖活动的迅猛发展以及养殖的不规范和不科学性导致近海生态系统结构和功能改变，一方面加重了海水富营养化，另一方面养殖动植物病害经常发生，严重影响了海产品的质量和效益。 大型海藻是海区重要的初级生产者，生命周期长、生长快，能通过光合作用吸收固定水体的C、N、P等营养物质来合成自身，同时增加水体溶解氧。因此，大型海藻被称为海洋环境中的生物过滤器。另外，由于大型藻类自身营养成分的复杂性和与藻共生的微生物多样性，大型海藻还可对生态系统中的浮游生物和微生物产生直接和间接影响。 在海洋环境，尤其是海水养殖水体环境存在着两个主要问题：海水富营养化和病源微生物控制，本文针对海洋环境中存在的这两个问题进行了探索研究。 以大型经济藻种长心卡帕藻（Kappaphycus alvarezzi）作为实验材料，分别在实验室内、室外藻类处理系统和海湾养殖现场三种条件下，进行藻类去除海水氮磷的一次性实验、半连续实验和连续实验，研究了其对海水中无机氮、无机磷的吸收速率和去除能力，初步评估了其生态价值。 构建了一种半封闭海域富营养化治理模式，以长心卡帕藻为实验材料，研究了其去除海水富营养化的能力，主要结果如下： （1）室内实验研究发现，长心卡帕藻对氮、磷的吸收速率随底物浓度升高而升高。在氮磷比为10:1，温度28℃条件下，氮浓度为50μmol • L-1时，藻对氮、磷的吸收速率达到最大，分别为0.93µmol • g-1（FW）• h-1和0.072µmol • g-1（FW）• h-1。 （2）人工修建的藻类养殖系统中进行的长心卡帕藻去除氮、磷的半连续实验，结果表明该藻具有连续去除海水DIN、DIP的能力。只要保持足够的底物浓度，长心卡帕藻对无机氮、无机磷的吸收速率达到最大，分别为0.3µmol • g-1（FW） • h-1和0.03µmol • g-1（FW）• h-1。但是对氮磷的吸收速率较室内实验有所降低。 （3）自然条件下，通过调查黎安海湾水质情况发现，长心卡帕藻具有较大的生态效益。在整个海湾大面积养殖卡帕藻，通过收获藻体，每年大约可以从海水中带走33吨氮素，7.5吨磷素。由于在海湾长心卡帕藻的作用，全年海湾水质保持在1-2级国家海水质量标准，产生了明显的生态效益。 另外，我们对大型藻类浒苔（Ulva clathrata）吸收氮磷和抑制鳗弧菌（Vibrio anguillarum）的效果进行了初步探索，结果表明：浒苔不仅对培养系统内无机氮和磷具有明显的去除作用，而且在异养细菌总量没有降低的情况下，对鳗弧菌有显著抑制作用，该抑制作用还受到水体中氮磷营养盐浓度的影响。在10g • L-1海藻的条件下，鳗弧菌以105－107 cfu • mL-1接入2天后，无论是否添加外源氮磷，鳗弧菌密度降到10 cfu • mL-1以下，鳗弧菌去除率几乎达到100%。实验数据还显示，添加氮磷营养盐可以增强浒苔对鳗弧菌的抑制作用，但没有降低其中的异养菌群数量，系统内异养细菌总量均维持在较高水平。进一步研究表明，培养浒苔24h后的海水，也对鳗弧菌65#产生抑制作用，这说明浒苔代谢释放到水体中某种化学成分或与藻共栖的微生物对鳗弧菌生长产生了抑制。

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。

中国明对虾Toll样受体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。自1993 年对虾白斑病暴发以来，中国明对虾的养殖一直一蹶不振。引起对虾大规模死亡的原因是多方面的，其主要原因是养殖环境恶化、对虾种质退化和抗病力下降。因此，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，改良种质和培育抗病品系，已成为对虾养殖业走可持续发展之路的当务之急。 Toll 样受体（Toll-like receptors, TLRs）家族是进化保守的哺乳动物模式识别蛋白（pattern recognition receptors, PRR），在先天免疫系统中起着非常重要的作用。本研究采用同源克隆和RACE（rapid amplification of cDNA ends）技术从中国明对虾中克隆到Toll 样受体同源基因，并将其命名为FcToll。它全长4115 bp，3’UTR 包含16 个poly A 尾巴，开放阅读框编码931 个氨基酸的多肽。预测的该多肽包含典型的Toll 样受体结构，分为胞外区、跨膜区和胞内区。其中胞外区有信号肽，有16 个富含亮氨酸的重复序列eucine-rich repeats, LRR），并含有2个LRR-C 末端基序和2 个LRR-N 末端基序；跨膜区是23 个氨基酸的一次跨膜结构域；胞内区是含有139 个氨基酸的TIR 结构域（Toll/Interleukin-1R）。克隆 发现FcToll 的基因组结构包含5 个外显子和4 个内含子。系统发生分析揭示FcToll归属于“昆虫型”的无脊椎动物Toll 样受体家族。组织分布研究发现FcToll 在中国明对虾中是组成型表达的，在淋巴器官中表达量较显著。分别利用不同病原体刺激健康的中国明对虾，Real-time PCR 发现该基因在刺激后表达水平呈现不同的表达谱：灭活鳗弧菌（Vibrio anguillarum）注射后5 小时，该基因表达显著 上调；而WSSV（white spot syndrome virus）注射后该基因表达则迅速下调，感染后23 小时内其表达水平均低于对应时间点的对照组。这就表明FcToll 可能参与中国明对虾的先天免疫防御，尤其可能参与入侵弧菌的免疫应答。

Cloning, characterization, and expression analysis of extracellular copper/zinc superoxide dismutase gene from bay scallop Argopecten irradians

Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned a novel ECSOD from the bay scallop Argopecten irradians (AiECSOD) by 3' and 5' RACE. The full-length cDNA of AiECSOD was 893 bp with a 657 bp open reading frame encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids, and sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms. The genomic length of the AiECSOD gene was about 5276 bp containing five exons and six introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NF kappa B, GATA-1, AP-1, and Ubx binding sites. Furthermore, tissue-specific expressions of AiECSOD and temporal expressions of AiECSOD in haemocytes of bay scallops challenged with bacteria Vibrio anguillarum were quantified using qRT-PCR. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12 h post-injection with V. anguillarum and then returned to normal between 24 h and 48 h post-injection. These results indicated that AiECSOD was an inducible protein and that it may play an important role in the immune responses against V anguillarum. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: Cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207 bp with a 678 bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V anguillarum infection. (c) 2008 Elsevier Ltd. All rights reserved.

Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai)

Heat shock protein 70 (HSP70), the primary member of HSPs that are responsive of thermal stress, is found in all multicellular organisms and functions mostly as molecular chaperon. The inducible HSP70 cDNA cloned from Pacific abalone (Haliotis discus hannai) using rapid amplification of cDNA ends (RACE), was highly homologous to other HSP70 genes. The full-length cDNA of the Pacific abalone HSP70 was 2631 bp, consisting of a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 573 by with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1968 bp. The HSP70 cDNA encoded a polypeptide of 655 amino acids with an ATPase domain of 382 amino acids, the substrate peptide binding domain of 161 amino acids and a C-terminus domain of 112 amino acids. The temporal expression of HSP70 was measured by semi-quantitative RT-PCR after heat shock and bacterial challenge. Challenge of Pacific abalone with heat shock or the pathogenic bacteria Vibrio anguillarum resulted in a dramatic increase in the expression of HSP70 mRNA level in muscle, followed by a recovery to normal level after 96 h. Unlike the muscle, the levels of HSP70 expression in gills reached the top at 12 h and maintained a relatively high level compared with the control after thermal and bacterial challenge. The upregulated mRNA expression of HSP70 in the abalone following heat shock and infection response indicates that the HSP70 gene is inducible and involved in immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Detection of serum and mucosal antibody production and antibody secreting cells (ASCs) in large yellow croaker (Pseudosciaena crocea) following vaccination with Vibrio harveyi via different routes

The kinetics of mucosal and serum antibody response is well as antibody secreting cells (ASCs) production were studied in large yellow croaker following vaccination with inactivated Vibrio harveyi by different routes: oral administration. intraperitoneal (IP) injection and immersion. Indirect ELISA was used to measure the antibody level in serum and cutaneous mucus, and ELISPOT was used to monitor the ASCs derived from gill, blood and head kidney. The data demonstrated that IP injection resulted in the highest antibody levels in the systemic circulation, whereas immersion induced significant antibody levels in mucous. As for the ASCs response, IP injection induced high numbers of ASCs in the head kidney and blood; oral intubation only induced a slight ASCs response in the head kidney: immersion induced a much stronger ASCs response in the gill. These results indicate that mucosal antibodies following immersion immunization are independent of a systemic response and more sensitive, since it could be triggered earlier than serum antibodies. The mucosal antibodies following IP injection immunization may depend oil a systemic immune response. The protective effects of the three vaccination methods were compared by challenging with live V. harveyi. Survival of the three groups of vaccinated fish varied front 40 to 60%. while 100% mortality was found in control fish. Compared with IP and oral vaccination, immersion stimulated higher specific antibody titers in the mucosal system and achieved similar protection, so it is in effective and efficient method for immunizing a large number of fish against V harveyi (C) 2008 Elsevier B.V. All rights reserved.

Optimization by orthogonal array design and humoral immunity of the bivalent vaccine against Aeromonas hydrophila and Vibrio fluvialis infection in crucian carp (Carassius auratus L.)

Aeromonas hydrophila and Vibrio fluvialis are the causative agents of a serious haemorrhagic septicaemia that affects a wide range of freshwater fish in China. In order to develop a bivalent anti-A. hydrophila and anti-V. fluvialis formalin-killed vaccine to prevent this disease, an orthogonal array design (OAD) method was used to optimize the production conditions, using three factors, each having three levels. The effects of these factors and levels on the relative per cent survival for crucian carp were quantitatively evaluated by analysis of variance. The final optimized formulation was established. The data showed that inactivation temperature had a significant effect on the potency of vaccine, but formalin concentration did not. The bivalent vaccine could elicit a strong humoral response in crucian carp (Carassius auratus L.) against both A. hydrophila and V. fluvialis simultaneously, which peaked at 3 or 5 weeks respectively. Antibody titres remained high until week 12, the end of the experiment, after a single intraperitoneal injection. The verification experiment confirmed that an optimized preparation could provide protection for fish at least against A. hydrophila infection, and did perform better than the non-optimized vaccine judged by the antibody levels and protection rate, suggesting that OAD is of value in the development of improved vaccine formulations.

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

Aims: To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins. Methods and Results: A gene (vhhP2) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24 V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non-V. harveyi species, including V. parahaemolyticus and V. alginolyticus. A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2. This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii, which is most closely related to V. harveyi. One of the V. campbellii strains was falsely identified as V. harveyi. Conclusions: vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non-V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi. However, this method can not distinguish some V. campbellii strains from V. harveyi. Significance and Impact of the Study: the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.

Characterization of degQ(Vh), a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50 C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E.coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

Characterization of degQ(Vh), a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain

Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50 C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E.coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

Cloning, characterization, and molecular application of a beta-agarase gene from Vibrio sp strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40 degrees C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.