Studies on the Pathogenic Bacterium and the DNA vaccine of Ulcer Disease in cage-cultured Epinephelus awoara

弧菌病是海水鱼类养殖业中造成经济损失最严重的细菌性疾病，本论文致力于养殖石斑鱼弧菌病病原菌及DNA免疫防治的研究，主要结果如下：1、调查研究了2002年夏季厦门同安湾网箱养殖石斑鱼大面积暴发的溃疡病，对细菌病原进行分离纯化，编号为TS-628，回归感染证实这株菌是引发同安湾网箱养殖石斑鱼溃疡病的病原菌。采用生化鉴定的方法对病原菌进行初步鉴定，同时采用PCR技术获得病原菌16SrRNA基因一段长1121bp的序列，该序列登录基因库（序列号为AY747308）。结合生化鉴定结果和16SrRNA基因同源性比较结果，确认分离到的病原菌为哈维氏弧菌(Vibrioharveyi)。42种药物的药敏实验结果...Vibriosis is the most serious bacterial disease that economically affects marine fish culture. In this thesis, an attempt had been made to study the pathogenic bacteria of vibriosis in cultured grouper and the feasibility of DNA vaccine technology as a prophylactic treatment to prevent the vibriosis. The results were shown as follows:1、The characteristics of ulcer disease occurred in cage-cultured...学位：理学博士院系专业：海洋与环境学院海洋学系_海洋生物学学号：B20022700

Study on protease activities of the ECP of pathogenic bacterium Vibrio harveyi strain TS-628

采用复性电泳技术,研究环境pH、温度及培养时间对哈维氏弧菌TS-628菌株胞外产物(ECP)蛋白酶活性的影响。结果表明,胞外产物蛋白酶最适反应pH在8.5左右,酸对ECP蛋白酶活性的抑制明显强于碱对它的抑制作用;最适反应温度在20～50℃之间,20℃以下或50℃以上蛋白酶活性则受到明显抑制,表明蛋白酶不仅对高温敏感,对低温也很敏感;培养12h的胞外产物蛋白酶活性最强。最适温度和pH条件下,主要出现3种蛋白酶,其中分子量为94kDa和26kDa的两种蛋白显示了很强的蛋白酶活性,而且能在比较极端的温度和pH值条件下保持活性,而分子量为35kDa蛋白酶只在最适反应条件下出现,并且该蛋白酶的最适反应条件与弧菌病暴发时的环境条件极为相似。由此可推测分子量94kDa和分子量为26kDa的两种蛋白可能是弧菌维持生存所必需,而分子量为35kDa的蛋白酶虽然只出现在特定条件下,却可能在致病过程中起重要作用。SDS-gelatin-PAGE assay was used to study the effects of pH,temperatures,and growing time on the protease activities of the extracellular products(ECP) of Vibrio harveyi TS-628 strain.The results showed that the optimal pH for ECP protease activity was about 8.5 and the protease activity was inhibited more easily by lower values of pH than by higher ones.The optimal temperature for the protease was 20~50℃,and significant decrease in activity was observed at temperatures below 20℃ or above 50℃,which indicated that the protease was sensitive to both low and high temperatures.The highest level of the extracellular product protease activity was present after 12h incubation.Under the condition of optimal temperatures and pH,there were three kinds of protease present:94kDa,26kDa and 35kDa.The 94kDa and 26kDa proteases displayed high activity and could remain active under extreme temperatures and pH,while the 35kDa protease was only active under the optimal condition,which was similar to the condition for the outburst of pathogenic vibrio.It is suggested that the 94kDa and 26kDa proteases were necessary in maintaining the vibrio survival and the 35kDa protease could play an important role in the pathogenicity of the bacteria.福建省青年科技人才创新基金项目(2006F3096);; 集美大学科研基金项目共同资

Establishment of in vitro biofilm model and characteristics of biofilm formation of Vibrio harveyi

采用改良微孔板法建立哈维氏弧菌VIbrIO HArVEyI TS-628菌株生物被膜体外模型,并进一步研究环境因子对哈维氏弧菌体外形成生物被膜的影响。结果发现,哈维氏弧菌TS-628菌株可以在聚苯乙烯板中形成生物被膜,并且该菌生物被膜的形成受起始菌液浓度、温度、PH等理化因子的显著影响。温度为30℃、nACl质量分数为3%—6%、PH为7、孵育时间为36H是哈维氏弧菌形成生物被膜的最佳条件。此外哈维氏弧菌在分别经表皮黏液、鳃黏液、肠道黏液、肝脏提取液、脾脏提取液、肌肉提取液包被的基质上成膜效果显著,其中在经肝脏提取液包被的基质上形成的生物被膜量显著高于在其他生物基质上形成的生物被膜量(P<0.01)。Modified microtiter-plate test was used to establish an in vitro biofilm model of Vibrio harveyi TS-628 strain.Effect of environmental factors on bacterial biofilm formation in vitro was also studied.The results show that V.harveyi TS-628 strains could form biofilm in polystyrene plates,and biofilm formation of this bacterium was significantly affected by different environmental factors,such as temperature,initial bacterial concentration,pH,NaCl concentration,and so on.The optimum conditions for biofilm formation of V.harveyi TS-628 strain are at temperature of 30℃,pH=7,NaCl concentration of 3%?6%,and using 36h incubation time.Furthermore,V.harveyi TS-628 strain could form biofilm on the walls coated with skin mucus,gill mucus,intestinal mucus,liver extract,spleen extract,and muscle extract.The biofilm formation of V.harveyi on the walls coated with liver extract exhibited a remarkably higher value of D(590)(p<0.01)than that on the walls coated with other tissue extracts.国家高技术研究发展计划项目(2007AA09Z115

The pathogeny of ulcer disease in Epinephlus awoara

2002年夏季厦门同安湾一带气候炎热,降雨量少,海水盐度偏高,同安湾刘五店的网箱养殖石斑鱼大面积暴发溃疡病。本研究调查了石斑鱼溃疡病暴发的特征和症状,主要表现为肌肉溃疡坏死、眼球脱落、鱼骨暴露等。从病鱼体表及内脏分离出优势菌群命名为TS 628,经回归感染证实TS 628就是引发本次石斑鱼溃疡病的病原菌。对病原菌进行鉴定发现,该菌革兰氏染色呈阴性,电镜下观察菌体呈短杆状,极端单鞭毛,综合研究该菌在形态、生理生化、16SrDNA同源性及药物敏感性等方面的特性,基本确认分离到的病原菌为哈维氏弧菌(Vibrioharveyi),该菌对氯霉素、壮观霉素等多种抗生素敏感,对万古霉素、青霉素G等抗生素不敏感。哈维氏弧菌是水产养殖病害常见病原菌,但作为养殖石斑鱼的病原菌在国内属首次报道。The summer of 2002 was hot and dry in Xiamen, and the seawater salinity was higher than usual, sometimes reaching 38 in Tongan Bay. From the end of May, ulcer disease occured on a large scale in Tongan Bay. In the middle of June, a heavy rain brought about high mortality among the cultured groupers, which caused serious loss. The characteristics of ulcer disease were observed. Infected groupers displayed sluggish swimming and refused feeding, several days later, the groupers' eyes swelled and became ulcerated, the tail turned red, the scales became detached and the back muscle gradually ulcerated, then the eyes even dropped out and the muscle became so necrotic that the spine was exposed, finally the diseased fishes died. Anatomized the dead fish and found that the livers and gills were pale and the gallbladder was distended. The time course from appearance of disease signs to death lasted about a week. Dominant bacteria, which were Gram-negative and seen short rod with single polar flagellum under electron microscope, were isolated and designated TS-628. In artifical infection test all fish　of the experimental group died on the third day after injected with bacterial suspension, while all the fish in control group showed no signs till the end of another week observation. The dead grouper had pale livers and gills and small ulcerations on the caudal fin and anal fins. These were the same signs as the natural infected fishes. The re-isolate also had the same characteristics as TS-628, which proved the isolate TS-628 was the pathogenic bacteria that triggered this ulcer disease. Different methods were used to identify the pathogenic bacteria in this study. The identification result through VITEK-AMS system GNI card was that TS-628 was Burkholderia mallei, with 99% reliability. While traditional biochemical identification revealed that TS-628 exhibited relVibrio harveyi through comparison with Bergey's Manual Determinative Bacteriology. In order to confirm absolutely different results above, further researches were carried out to identify TS-628 again. So 471bp sequence of TS-62816S rRNA gene was amplify and compared with all DNA sequences (1192858 in total) in genebank (GenBank+EMBL+DDBJ+PDB), homology analyses showed that 16 sequences were picked out to have the highest similarity, with 98% identity. These 16 sequences all originated fromV. harveyV. carchiariae which also belongs to harveyi because their similarity in physiological and biological characteristics and DNA homology. According to morphological features, physiological and biochemical characteristics and 16S rRNA gene homology comparison of the bacteria, the patV. harveyi. Drug sensitivity test showed that the pathogenic bacteria were highly sensitive to chloramphenicol, actinospectcinV. harveyi is a kind of pathogenic bacterium commonly found iV. harveyi is reported as the pathogenic bacteria of cage-cultured grouper in China. And V. harveyi should be regarded as an opportunistic pathogen which has close relation to temperature and salinity and easily causes vibriosis under conditions of high temperatures and drought. Therefore, it is necessary to guard agV. harveyi vibriosis in such summer days.福建省重大科技项目资助(2002N009

Antigenicity Analysis of V.harveyi TS-628 Strain

哈维氏弧菌(Vibrio harveyi)是海水鱼虾养殖中常见的致病菌,由该菌引发的病害给世界各地的养殖业带来重大经济损失,但对其有效抗原的筛选或相关疫苗的研究报道相当少,对其病害的防治也尚无有效措施.本文以患病青石斑鱼分离到的病原菌哈维氏弧菌TS-628菌株为研究对象,分别提取它的鞭毛蛋白、脂多糖(LPS)和外膜蛋白(OMP),并采用Western blot技术分析检测这3种成分的抗原性.结果显示,鞭毛蛋白主要的免疫印迹带约有4条,其中435、2 ku为主要免疫反应显色带;OMP主要的免疫印迹带约有5条,其中43 ku为最主要免疫反应显色带,35、38、47和52 ku也具有较强的免疫显色反应.LPS没有检测到免疫印迹反应.这一研究结果将为灵敏检测哈维氏弧菌以及研制高效疫苗奠定基础.Vibrio harveyi,the major causal agent of vibriosis,affects a diverse range of marine cultured organisms over a wide geographical area,while the reports about screening the effective antigen and the researches on vaccines of V.harveyi are scarce.Flagellin,lipopolysaccharide(LPS) and outer membrane proteins(OMP) are major immunogenic antigens in many Gram-negative bacteria.In this study,the flagellin,OMP and LPS of V.harveyi TS-628 strain isolated from the groupers infected by it were extracted and Western blot analysis was used to detect the antigenicity of these extractions.The results of the Western blot assay revealed that there were 4 positive flagellin bands about 35,38,43 and 52 ku,of which the 43 and 52 ku bands displayed the strongest positive reaction.There were 5 positive OMP bands about 35,38,43,47 and 52 ku,of which the 43 ku appeared the strongest positive reaction and the other four proteins also displayed strong reaction.However the LPS was Western blot-negative.These results indicated that the 43 and 52 ku flagellin and the OMP such as 43,52 ku proteins could be the candidates to develope vaccines against V.harveyi.国家“863”计划(2003AA603011);; 福建省科技项目(2002N009)资

Genetic diversity of Sipunculus nudus along the coasts of China

为科学保护和开发我国光裸方格星虫自然资源,本研究以cytb基因全长为分子标记分析了山东烟台、福建古雷、香港、海南儋州、海南陵水、广西东兴6个光裸; 方格星虫群体的遗传多样性。结果显示,203个个体共检出128个单倍型,299个多态位点,核苷酸多样性和单倍型多样性分别为0.0488和0.977; 3,且变异主要来自群体间(88.4%)。6个光裸方格星虫群体间遗传分化指数(F_(st))分析结果显示,南方5个群体间F_(st)0.15,群体间呈高度分化。中性检验(Fu's; Fs、Tajima's; D)结果为负值,北方群体和南方群体的核苷酸错配分布图为单峰,单倍型网络分布图为以主单倍型为中心的放射状结构。用203条cytb序列构建的系统发育; 树显示,烟台群体聚为一个进化支,南方群体聚为一个进化支。研究表明,中国沿海的光裸方格星虫群体在历史上经历过大规模群体扩张,但目前遗传多样性较低,; 说明光裸方格星虫对环境变化的适应能力较差,应加强野生资源的保护,在进化关系上,烟台群体与其他5个群体之间存在较大的遗传分化,但是否已经形成隐秘种; 仍需进一步的研究。Sipunculus nudus resources have been seriously damaged in the recent; years due to overfishing and habitat destruction. Protecting natural; resources of S. nudus has become more and more urgent. In this paper,; genetic structure and population differentiation of S. nudus from 6; localities along the coasts of China were analyzed based on sequences of; mitochondrial cytochrome b gene. Complete mitochondrial cytb sequences; from 203 individuals of S. nudus were analyzed, 299 polymorphic sites; and 128 haplotypes were identified and the nucleotide diversity and; haplotypes diversity were 0.0488 and 0.9773, respectively. The variation; among populations (accounting for 88.4%) was the main source of; variation. According to differentiation index (F_(st)) in populations of; S. nudus, the 5 populations in the south of Yantai showed no significant; differences or low differences, with F_(st)0.15.; Fu's Fs and Tajima's D were negative. Figures of mismatch distribution; were unimodal. Haplotype network was radial structure centering on main; haplotype. NJ phylogenetic tree of S. nudus based on 203 sequences of; cytb of mtDNA revealed that these 6 populations were divided into 2; clades, one clade was Yantai population and the other clade included the; rest 5 populations in the south of Yantai. All these results revealed; that S. nudus population had experienced a large-scale population; expansion in history, and the relatively low genetic diversity of S.; nudus now suggested that the ability of S. nudus to adapt to; environmental changes was poor and some measures should be taken to; protect S. nudus resources. Phylogenetic tree suggested Yantai; population was distinct from the other 5 populations, but whether Yantai; population has become a cryptic species still needs further research.国家自然科学基金; 海洋公益性行业科研专项; 海洋经济创新发展区域示范项

Cloning and sequencing of FlaA gene of Vibrio harveyi and construction of its eukaryotic expression recombinant plasmid

哈维氏弧菌是水产病害中常见的致病菌.参照基因库上登录的弧菌鞭毛丝蛋白FlaA基因序列设计简并引物,PCR扩增哈维氏弧菌TS-628株的FlaA全基因,并克隆到pMD 18-T载体中测序,经序列分析该基因全长1 140 bp,编码379个氨基酸.与基因库中其他弧菌的同源基因序列比较显示,哈氏弧菌FlaA基因与霍乱弧菌FlaA基因的同源性最高(79.5%),该基因编码的多肽缺乏半胱氨酸,并在N-,C-两端的氨基酸序列较为保守,中间区域的变异较大.对该蛋白的氨基酸组成及空间结构进行了分析和预测,并推测该蛋白质的平均分子量为40.6 kDa.在该基因末端加上一段编码Flag短肽的核苷酸序列后克隆到真核表达载体pcDNA3.1(+),获得带哈氏弧菌鞭毛丝蛋白FlaA基因的真核表达重组质粒pcDNA-FlaA-tag,为其DNA疫苗的进一步研究奠定了基础.Vibrio harveyi is a kind of conditioned pathogen often found in the marine fishery aquaculture.In this study,a pair of degenerate primers were designed according to the homologous sequences of the FlaA genes from Genbank to amplify the FlaA gene of V.harveyi TS-628 strain,and the suitable PCR product was cloned into a pMD 18-T vector.After sequencing and analyzing,the FlaA gene was found to contain 1 140 bp,which encodes 379 deduced amino acids.Presumably the FlaA gene would encode a protein of 40.6 kDa based on DNA-deduced amino acid sequence.When compared with homologs in other vibrios from Genbank,the FlaA gene of V.harveyi revealed the greatest homology with that of V.cholerae(79.5%).The comparison also indicated that these polypeptides were typically conserved in amino-and carboxy-termini,while the central regions were more diverse and there were no cysteine residues in the flagellin.Then a pair of specific primers,with a short nucleotide sequence encoding Flag tag,were designed according to the obtained sequence of the FlaA genes to amplify the FlaA of V.harveyi.The PCR product was cloned into eukaryotic expression vector pcDNA3.1(+)and the positive clone was chosen and identified through the digestion analysis and sequencing.A eukaryotic expression recombinant plasmid,pcDNA-FlaA-tag,containing the FlaA gene of polar flagellin in V.harveyi was constructed,which would be a foundation for further study on its DNA vaccine.国家863项目(2003AA603011);; 福建省重大科技项目(2002N009

STUDY ON THE KARYOTYPE IN Nibea miichtheoides

采用体内注射PHA和染色体常规制片方法对状黄姑鱼 (NibeamiichtheoidesChu,Lo &Wu)的核型进行了研究 ,结果表明状黄姑鱼二倍体染色体数目为2n=48,核型为2n=48t ,臂数NF=48。本文还就鱼类的染色体核型演化趋势进行了探讨。The karyotypes of Nibea miichtheoides collected from Xiamen in Fujian and Zelin in Guangdong in Apr. 2000 and Jan. 2001 respectively were analyzed by injection of PHA and colchicines in this paper. Under the microscopes ,the chromosomes prepared from the head-kidney tissues were observed and measured. The result showed that the karyotype of Nibea miichtheoides was 2 n=48 t, NF=48. The evolution trend of fish karyotypes was also discussed in this paper.国家自然科学基金资助项目39870562号

The karyotypes of Plectorhinchus cinctus

1 引言 一位著名的遗传学家说过,地球的历史记载于地壳中,而生物的历史却记载在染色体上,染色体作为遗传信息的载体具有种的特异性,不同的物种有其特定的染色体组型,在很大程度上染色体的组型反映了物种的进化历史和种间的亲缘关系。因此,染色体组型研究成为细胞遗传学上一个不可缺少的部分,对种质资源的研究来说,更具有极其重要的意义。本文以我国沿海常见的经济鱼类花尾胡椒鲷为研究对象,分析了它的组型

副溶血弧菌(Vibrio parahaemolyticus)极鞭毛蛋白FlaB基因的克隆与序列分析

鱼类已经具有特异性免疫系统,在抗原的刺激下能产生 IgM 类免疫球蛋白,因此利用疫苗对鱼类病害进行免疫防治是比较理想的途径。DNA 疫苗是将含有编码保护性抗原基因的真核表达质粒直接接种到宿主体内,被体细胞摄取并表达相应抗原蛋白,以刺激机体产生相对应的免疫应答,从而达到免疫防治的目的。DNA 疫苗的免疫原性和免疫保护作用的有效性也已经在大范围的动物模型和初步的人体临床实验中得了证明。本实验室用人工合成的方法克隆了极鞭毛蛋白 Fla I 基因,并尝试在 COS-7细胞中进行表达,证