Molecular characterizations of oxytetracycline resistant bacteria and their resistance genes from mariculture waters of China

Oxytetracycline-resistant bacteria were isolated from a mariculture farm in China, and accounted for 32.23% and 5.63% of the total culturable microbes of the sea cucumber and the sea urchin rearing waters respectively. Marine vibrios, especially strains related to Vibrio splendidus or V. tasmaniensis, were the most abundant resistant isolates. For oxytetracycline resistance, tet(A), tet(B) and tet(D) genes were detected in both sea cucumber and sea urchin rearing ponds. The dominant resistance type for V. tasmaniensis-like strains was the combination of both tet(A) and tet(B) genes, while the major resistance type for V. splendidus-like strains was a single tet(D) gene. Most of the sea cucumber tet-positive isolates harbored a chloramphenicol-resistance gene, either cat IV or cat II, while only a few sea urchin tet-positive isolates harbored a cat gene, actually cat IV. The coexistence of tet and cat genes in the strains isolated from the mariculture farm studied was helpful in explaining some of the multi-resistance mechanisms. (c) 2006 Elsevier Ltd. All rights reserved.

Concurrence of cat and tet genes in multiple antibiotic-resistant bacteria isolated from a sea cucumber and sea urchin mariculture farm in China

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.

A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge

Rel/NF kappa B is a family of transcription factors. In the present study, a Rel/NF kappa B family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627 bp, revealed a 1071 bp open reading frame encoding 357 aa. The predicted molecular weight (MW)of the deduced amino acid sequence of FcDorsal was 39.78 kDa, and its theoretical pl was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G(+)) bacteria (Micrococcus lysodeikticus) and Gram-negative (G(-)) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NF kappa B member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819)

Serine protease inhibitors, critical regulators of endogenous proteases, are found in all multicellular organisms and play crucial roles in host physiological and immunological effector mechanisms. The first mollusk serine proteinase inhibitor (designated AISPI) cDNA was obtained from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the scallop serine protease inhibitor was 1020 bp, consisting of a 5'-terminal untranslated region (UTR) of 39 bp, a 3'-terminal UTR of 147 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 834 bp. The AISPI cDNA encoded a polypeptide of 278 amino acids with a putative signal peptide of 22 amino acids and a mature protein of 256 amino acids. The deduced amino-acid sequence of AISPI contained six tandem and homologous domains similar to that of Kazal-type serine protease inhibitors, including the conserved sequence C-X(7)-C-X(6)-Y-X(3)-C-X(2,3)-C and six cysteine residues responsible for the formation of disulfide bridges, indicating that the AISPI protein from bay scallop should be a member of the Kazal-type serine protease inhibitor family. The temporal expression of AISPI was measured by semi-quantitative RT-PCR after injury or bacterial challenge. After the adductor muscle was wounded or injected with Vibrio anguillarum, the expression of AISPI mRNA in hemolymph was up-regulated and reached the maximum level at 8 and 16 h, respectively, and then progressively dropped back to the original level. The results indicated that AISPI could play an important role in injury healing and immune response in mollusks as it could be induced by injury and bacterial challenge. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular characterizations of chloramphenicol- and oxytetracycline-resistant bacteria and resistance genes in mariculture waters of China

In order to gain an understanding of the diversity and distribution of antimicrobial-resistant bacteria and their resistance genes in maricultural environments, multidrug-resistant bacteria were screened for the rearing waters from a mariculture farm of China. Both abalone Haliotis discus hannai and turbot Scophthalmus maximus rearing waters were populated with abundant chloramphenicol-resistant bacteria. These bacteria were also multidrug resistant, with Vibrio splendidus and Vibrio tasmaniensis being the most predominant species. The chloramphenicol-resistance gene cat II, cat IV or floR could be detected in most of the multidrug-resistant isolates, and the oxytetracycline-resistance gene tet(B), tet(D), tet(E) or tet(M) could also be detected for most of the isolates. Coexistence of chloramphenicol- and oxytetracycline-resistance genes partially explains the molecular mechanism of multidrug resistance in the studied maricultural environments. Comparative studies with different antimicrobial agents as the starting isolation reagents may help detect a wider diversity of the antimicrobial-resistant bacteria and their resistance genes. (C) 2009 Elsevier Ltd. All rights reserved.

Diversity of Both the Cultivable Protease-Producing Bacteria and Their Extracellular Proteases in the Sediments of the South China Sea

Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

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。

织纹螺（Nassarius spp.）体内河豚毒素来源的研究

近几十年来，国内沿海地区频繁发生食用织纹螺中毒事件，并导致数十人死亡，这一问题得到了政府相关部门的高度重视。但是，由于织纹螺毒性变化很大，毒素来源不清楚，因此很难预测食用织纹螺中毒事件的发生，这在很大程度上限制了对食用织纹螺中毒事件的有效监测和管理。目前，对于中国沿海有毒织纹螺体内河豚毒素（tetrodotoxin, TTX）的来源还未见过系统研究。本文选取中国沿海常见的半褶织纹螺（Nassarius semiplicatus）、纵肋织纹螺（N. variciferus）和拟半褶织纹螺（N. semiplicatoides sp. nov.）作为实验对象，从毒素的微生物来源与食物链来源这两个角度分别展开研究，以探讨织纹螺体内 TTX 的可能来源，为提出相应的预防管理措施提供科学依据。 首先，我们先后从曾发生过中毒事件的江苏盐城和连云港采集了织纹螺样品，通过小鼠生物测试法和液-质联用分析技术（LC-MS），对织纹螺的毒性和毒素组成进行了测试和分析，分离培养了织纹螺体内及其生活环境中的细菌，应用河豚毒素单克隆抗体酶联免疫检测方法（ELISA）对细菌的产毒情况进行了测试，并通过 16S 核糖体（rRNA）部分基因序列测定对细菌种类进行了初步的分析。研究发现，采自江苏盐城和连云港的半褶织纹螺的毒性分别约为 2 MU/g 和 200 MU/g 组织，体内的毒素成分是河豚毒素及其同系物。从盐城的半褶织纹螺及其生活环境分离的菌株中随机挑出 14 个菌株中，9 个菌株河豚毒素检测结果呈现阳性。从连云港高毒性半褶织纹螺消化腺中分离到的 45 个菌株中，阳性菌株有 21 个。但是，有毒菌株毒素含量较低，毒素含量范围是 15-184ng/g。通过 16S rDNA 部分序列的测序结果发现，大部分有毒菌株与弧菌属（Vibrio）的细菌在遗传序列信息上比较相近。其余有毒菌株分别与希瓦氏菌属（Shewanella）、海单胞菌属（Marinomonas）、黄杆菌属（Tenacibaculum）、动性菌属（Planococcus）、发光杆菌属 (Photobacterium)和气单胞菌属（Aeromonas）的遗传序列比较相近。其中与海单胞菌属、动性菌属和发光杆菌属亲缘关系较近的产毒细菌是首次报道。这一研究表明织纹螺体内及其生活环境中的存在产河豚毒素的细菌，但由于产毒素的量较低，因此可能在织纹螺体内河豚毒素的产生和累积过程并不发挥主要作用。 织纹螺作为一类腐食性的海洋动物，也有可能通过进食含有河豚毒素的生物而累积河豚毒素。对此，我们开展了高毒性半褶织纹螺的室内培养实验，以及河豚毒素在不同种类织纹螺体内的累积和排出的模拟实验，并定期采样，通过液相色谱与串联质谱联用技术（LC-MS/MS）对织纹螺体内河豚毒素及其同系物的含量变化情况进行了分析。室内培养实验发现，从连云港赣榆县采集的高毒性半褶织纹螺，在实验初期，体内毒素含量呈下降的趋势，但从 7月上旬开始，毒素含量突然快速上升，与连云港赣榆县野外采集的织纹螺的毒素含量表现出相似的变化趋势。河豚毒素在不同种织纹螺体内的累积和排出的模拟实验发现，通过投喂高毒性的河豚鱼肝脏（毒性为5×103 MU/g），纵肋织纹螺在一段时间内能够快速累积少量的河豚毒素。当停止投喂有毒河豚鱼肝脏后，毒素含量会快速下降。而在曾导致中毒事件的拟半褶织纹螺中，投喂有毒河豚鱼的肝脏后，其体内毒素含量只有缓慢增加。但在投喂无毒的河豚鱼肝脏后，其毒性却出现了快速增加的现象，这与该地区野外样品的毒性变动状况类似。这些发现显示高毒性半褶、拟半褶织纹螺体内的河豚毒素应当不是食物链累积的结果，而可能是由其自身产生。并且，毒素含量的变化具有一定的生物节律，有可能与产卵、繁殖等自然节律相关。 通过对半褶、纵肋和拟半褶织纹螺的研究工作可以认为，产河豚毒素的细菌不是织纹螺体内河豚毒素的主要来源，并且毒素也不是来自其摄食的食物，推测可能主要是由织纹螺自身产生。织纹螺所表现出的河豚毒素含量的季节性变化，极有可能与产卵、繁殖等自然节律相关，这些发现为预防和管理食用织纹螺中毒事件提供了科学依据。但是，本研究并未完全阐明织纹螺体内河豚毒素的来源，对于织纹螺体内河豚毒素的确切来源以及河豚毒素的代谢和转化机制，还有待于更加深入地研究工作。

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。

海藻表面附着弧菌多样性分析及方法研究

对四种大型海藻：江篱(Gracilaria textorii)、孔石莼（Ulva pertusa）、海带苗（Laminaria japonica）和多管藻（Polysiphonia urceolata）表面附着弧菌的多样性进行了分析，同时对多样性分析中采用的不同方法进行了比较。 利用TCBS培养基从四种海藻表面总共分离到12株细菌：G1、G2分离自江篱，U3分离自孔石莼，L4、L5、L6分离自海带苗，P7、P8、P9、P10、P11、P12分离自多管藻。菌株G1，G2属于盐单胞菌(Halomonas)，其余10株细菌都属于弧菌(Vibrio)。 对12株细菌的酶学活性、抗生素抗性进行了研究。发现除G1、G2外，其余菌株都可产生淀粉酶和明胶酶。菌株G1、G2和U3对氨苄青霉素、链霉素和四环素有很强抗性。 采用多种分子生物学方法（16S rDNA、gyrB、RFLP、DGGE ）分析了上述12株菌的系统发育关系，对不同方法获得的结果进行了比较。 12株菌的16S rDNA系统发生树可分为4个明显分支。G1、G2与细菌H. meridiana构成一个分支。U3与V. lentus构成一个分支。L4、L5、L6、P9、P10、P11、P12与V. tasmaniesis形成一个分支。P7、P8与V. splendidus形成一个分支。 以gyrB基因序列为基础构建的系统发生树将G1、G2以外的10株弧菌分成4个较大分支，L4、P9属于同一分支，L6、P8、P10、P11和P12属于一个大的分支， U3、L5各自构成一个分支。菌株G1、G2没有得到gyrB基因序列扩增带。表明试验设计的引物是弧菌特异性引物。相对16S rDNA，不同菌株间gyrB基因序列差异性更大。 RFLP分析结果显示，12株细菌HinfI酶切后得到6种带型，SmaI酶切后得到4种带型。结合16S rDNA、gyrB基因的分析结果可知，RFLP可以在种的水平上有效进行细菌多样性分析。 12株细菌的DGGE分析结果显示，G1、G2与其它菌株电泳图谱有明显不同。菌株U3独自构成一种带型，L5、L6构成一种带型。表明DGGE技术在属的水平上分析细菌多样性效果较好，在种的水平上分析细菌多样性有一定的局限性。

弧菌琼胶酶和丝氨酸蛋白酶的筛选、分析以及免疫应用

从山东黄岛海水养殖场分离到一株弧菌V134，从中克隆得到琼胶酶基因agaV，并将其在大肠杆菌中表达，纯化得到重组的琼胶酶AgaV。酶活分析发现该琼胶酶的最适温度在40℃左右，对pH比较敏感，pH 7.0时具有最高的琼胶裂解活性。对AgaV进行了两种应用性探索：（1）利用AgaV从琼脂糖凝胶中回收DNA，回收效率可达90%以上；（2）利用agaV作为报告基因构建了捕获分泌序列的载体pBU，并用其从革兰氏阳性细菌（G+菌）和革兰氏阴性细菌（G-菌）中筛选出了一系列分泌蛋白。将利用pBU从一株哈维氏弧菌T4中筛选出的6个分泌蛋白分别进行基因克隆、蛋白表达纯化和牙鲆免疫实验，发现其中一个蛋白，命名为DegQVh，具有免疫保护效应，其免疫保护率（RPS）可达64%。为了提高DegQVh的免疫保护效应，将AgaV的分泌结构域与DegQVh融合，构成融合抗原AgaV－DegQVh。利用大肠杆菌作为载体菌构建了AgaV－DegQVh融合抗原递呈系统，用其作为疫苗进行免疫，发现其RPS可达到95%。酶活分析表明DegQVh在50℃、pH 8.0时具有最高的活性。突变分析表明83位的组氨酸、113位的天冬氨酸和188位的丝氨酸以及两个PDZ结构域是DegQVh活性所必需的。表达分析发现degQVh表达受温度和细胞浓度调控，并且其上游有一个受E调控的启动子。进一步的分析发现DegQVh能够与大肠杆菌的DegP功能互补。

哈氏弧菌种特异性分子标记的发现及其在哈氏弧菌快速诊断和免疫防治中的应用

哈氏弧菌是危害水产养殖业发展的重要病原菌之一，因而其免疫防治研究具有重要意义。论文发现了一个新的、编码未知功能蛋白的基因vhhP2，该基因只存在于哈氏弧菌中，具有很高的种特异性。根据此特点，建立了一种vhhP2 -PCR检测方法，并证明该方法可以快速准确地从动物血液、组织以及环境样品中检测哈氏弧菌。同时，对VhhP2进行了免疫原性检测，发现VhhP2作为亚单位疫苗具有良好的免疫保护效应。为了克服常规亚单位疫苗的缺点以及进一步提高VhhP2的免疫效应，利用表面展示技术将VhhP2蛋白定位到鱼类共生菌细胞表面，制成活体疫苗。免疫结果表明，这种表面展示型VhhP2能够大幅提高牙鲆对哈氏弧菌的抵抗能力。论文还利用VhhP2蛋白的分泌功能，将VhhP2与一株迟缓爱德华氏菌的免疫保护性抗原蛋白重组融合，制成交叉保护疫苗，并证明其对哈氏弧菌和迟缓爱德华氏菌均有一定的免疫作用。