鳗弧菌金属蛋白酶基因在大肠杆菌中的表达、纯化、定点突变及其DNA疫苗的研制
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摘要
鳗弧菌(Vibrio anguillarum)能引起世界范围内的淡水、海水鱼类及其它养殖动物发病,其病症为快速的爆发性败血症及组织损伤,病鱼在短期内死亡,常给水产养殖业造成巨大的经济损失。鳗弧菌W-1分离自山东莱州湾海区发病的鲈鱼,已有的研究表明,W-1胞外金属蛋白酶是其重要的毒力因子之一,在细菌致病过程中发挥了重要的作用。编码金属蛋白酶的empA基因已被克隆和测序。
本研究将鳗弧菌W-1金属蛋白酶基因empA分别克隆到原核表达载体pBV220、pBAD24和pET24d(+)中,得到的三种重组质粒分别转化大肠杆菌JM109、TOP10和BL21(DE3)。比较empA基因在三种重组菌JM109/pBV220/empA、TOP10/pBAD24/empA和BL21(DE3)/pET24d(+)/empA中的表达情况,最终选择重组菌BL21(DE3)/pET24d(+)/empA来表达、纯化及定点突变研究empA基因。对重组菌培养及诱导条件进行优化后发现,25℃时经1 mM IPTG诱导重组菌体表达的EmpA金属蛋白酶可溶性蛋白表达量最多、包涵体形成最少,并且分泌到培养上清液中的EmpA金属蛋白酶量也最大;Western-blot检测重组表达的EmpA金属蛋白酶具有抗原性。进一步用Ni亲合柱从重组菌菌体及其培养上清液中纯化了EmpA金属蛋白酶,SDS-PAGE电泳测得分子量都为36 kDa,而未经加热处理的蛋白分子量为44.6 kDa;用Sephadex G-200测得蛋白分子量都为44.6 kDa,表明在大肠杆菌中表达的EmpA金属蛋白酶为单体蛋白。纯化的EmpA金属蛋白酶对牙鲆鳃细胞系(FG)有毒性作用,用5μg的蛋白作用于FG,在12 h内即可观察到细胞的死亡;酶对大菱鲆也表现出致死毒性,其症状与感染鳗弧菌的病鱼症状相似,半致死剂量LD50为8.1μg/鱼。
利用PCR方法对推测的EmpA金属蛋白酶活性位点氨基酸的碱基序列进行了定点突变,将突变质粒转化到大肠杆菌BL21(DE3)中进行表达,并用Ni亲合柱分别纯化了各突变蛋白。比较EmpA金属蛋白酶及其突变蛋白的蛋白水解活性和细胞毒性发现,突变蛋白都不同程度地丧失了其蛋白水解活性和细胞毒性,表明突变位点的氨基酸对酶活性起着十分重要的作用。
将没有蛋白水解活性和细胞毒性的m-EmpA7蛋白的基因序列m-empA7克隆到真核表达载体pEGFP-N1中,得到真核重组表达质粒pEGFP-N1/m-empA7。用磷酸钙共沉淀法将重组质粒转染至CHO和HEK293T细胞中,通过荧光显微镜、RT-PCR检测和Western-blot检测证实目的基因在真核细胞内获得了表达。
对牙鲆肌肉注射50μg/尾的重组质粒pEGFP-N1/m-empA7,于接种后不同时间分别取对照组和试验组牙鲆各组织做冷冻切片,在荧光显微镜下观察可见,在试验组牙鲆的注射点周边肌肉、注射点对面体侧肌肉、脾脏、前肾、体肾、后肠、心、鳃和肝脏等部位均可见绿色荧光,对照组组织没有荧光。RT-PCR检测和Western-blot检测的结果也证实目的基因在牙鲆体内获得了表达。
为研究真核重组表达质粒pEGFP-N1/m-empA7对牙鲆的免疫效果,分别以5μg/尾、20μg/尾和50μg/尾的不同剂量对牙鲆进行肌肉注射免疫,同时设置注射PBS和空质粒的对照组。ELISA检测结果显示,各免疫组牙鲆体内均产生了抗m-EmpA7抗原的特异性抗体,且抗体水平随接种剂量的增加而升高;而对照组牙鲆血清基本没有抗体凝集发生,在不同时间内抗体水平无明显变化。免疫后第5周,用鳗弧菌W-1对各组进行攻毒实验,各免疫组的免疫保护率(RPS)分别为57.1%,71.4%和85.7%,与对照组相比,免疫组产生了显著的保护作用(p < 0.001)。
Vibrio anguillarum is the causative agent of vibrosis, one of the major bacterial disease affecting marine and freshwater fishes in the world. The infected fish showed a rapid fulminating hemorrhagic septicemia and wide-spread tissue damage, and died in several days. The distribution of vibrosis is worldwide, which cause great economic loss to the aquaculture industry. Vibrio anguillarum strain W-1 was originally isolated from diseased seabass (Lateolabrax japonicus). Previous studies revealed that the extracellular protease EmpA of Vibrio anguillarum was one its important virulence factors. The gene coding EmpA was cloned and sequenced.
In this study, empA was cloned into three prokaryotic expression plasmids pBV220, pBAD24 and pET24d(+) respectively, resulting three recombinant Escherichia coli strains JM109/pBV220/empA、TOP10/pBAD24/empA and BL21(DE3)/pET24d(+)/empA. We finally chose BL21(DE3)/pET24d(+)/empA to express, purify and mutate empA since comparisons of the expression of empA in this three recombinant strains showed that BL21(DE3)/pET24d(+)/empA had the best expression profile. The expression conditions of the recombinant E. coli BL21(DE3)/pET24d(+)/empA were optimized. It was found that the soluble productions of EmpA expressed in recombinant cells and secreted to the medium were both maximal when E. coli BL21(DE3)/pET24d(+)/empA was induced with 1 mM IPTG at 25℃. The expressed reombinant EmpA could react with the anti-EmpA serum in Western-blot. EmpAs expressed in recombinant E. coli and secreted to the medium were purified by metal chelating affinity chromatography. Estimated molecular weights of purified EmpAs were both 36 kDa by SDS-PAGE, but both 44.6 kDa if the samples were not heated in boiling water. And they were both determined to be 44.6 kDa by Sephadex G-200 gel filtration. Thus, EmpA expressed in E. coli was a monomer polypeptide. Purified EmpA had cytotoxicity to flounder gill cell line (FG). The development of CPE-like morphologic damage of the cells was visible by light microscopy within 12 h of incubation of the cells with 5μg of purified EmpA. It could also cause death of turbot when injected intraperitoneally, which showed similar symptoms with diseased fish infected by Vibrio anguillarum. The LD50 of EmpA to turbot was established as 8.1μg protein/g of fish.
The nucleotides of the amino acid residues in the active sites of EmpA were mutated by PCR site-directed mutagenesis. All recombinant plasmids with mutated empAs were transformed into E. coli BL21(DE3), and mutated EmpAs were expressed and purified to characterize the effects of alterations of these amino acid residues on enzymatic activity. It was found that all of the single point mutations at the conservative residues reduced the proteolytic activity and cytotoxicity of the enzyme with different levels, indicating that they play an important role in enzymatic activity.
To construct DNA vaccine, mutated empA (m-empA7) was subcloned into eukaryotic expression vector pEGFP-N1, resulting the recombinant plasmid pEGFP-N1/m-empA7. It was then transfected into CHO and HEK293T cells and the expression of m-empA7 was detected with fluorescent microscope, RT-PCR and Western-blot, which indicating that m-empA7 was expressed in CHO and HEK293T cells.
Flounder were intramuscularly injected with 50μg of the DNA vaccine solution to examine if m-empA7 could be expressed. Fluorescent microscope, RT-PCR and Western-blot detection revealed that m-empA7 was expressed in the muscle, spleen, kidney, hind intestines, heart, gill and liver of flounder.
To study the protective efficacy of the recombinant plasmid pEGFP-N1/m-empA7 on flounder, fish were respectively immunized with three doses of DNA vaccine (5μg/fish, 20μg /fish and 50μg /fish). The results of ELISA revealed that the immunized fish harvested much higher special antibody against m-EmpA7 in relation to negative controls of PBS buffer and pEGFP-N1, and the antibody titer increased as the immunized dose increased. Five weeks after vaccination, fish were challenged by intramuscularly injection of Vibrio anguillarum W-1 cell suspension. Mortalities following exposure to the bacteria were much lower in fish vaccinated with pEGFP-N1/m-empA7 compared to those of the control groups injected with PBS buffer and pEGFP-N1 alone. DNA vaccine of three doses protected fish from the bacterium with 57.1%, 71.4% and 85.7% of RPS respectively.
本研究将鳗弧菌W-1金属蛋白酶基因empA分别克隆到原核表达载体pBV220、pBAD24和pET24d(+)中,得到的三种重组质粒分别转化大肠杆菌JM109、TOP10和BL21(DE3)。比较empA基因在三种重组菌JM109/pBV220/empA、TOP10/pBAD24/empA和BL21(DE3)/pET24d(+)/empA中的表达情况,最终选择重组菌BL21(DE3)/pET24d(+)/empA来表达、纯化及定点突变研究empA基因。对重组菌培养及诱导条件进行优化后发现,25℃时经1 mM IPTG诱导重组菌体表达的EmpA金属蛋白酶可溶性蛋白表达量最多、包涵体形成最少,并且分泌到培养上清液中的EmpA金属蛋白酶量也最大;Western-blot检测重组表达的EmpA金属蛋白酶具有抗原性。进一步用Ni亲合柱从重组菌菌体及其培养上清液中纯化了EmpA金属蛋白酶,SDS-PAGE电泳测得分子量都为36 kDa,而未经加热处理的蛋白分子量为44.6 kDa;用Sephadex G-200测得蛋白分子量都为44.6 kDa,表明在大肠杆菌中表达的EmpA金属蛋白酶为单体蛋白。纯化的EmpA金属蛋白酶对牙鲆鳃细胞系(FG)有毒性作用,用5μg的蛋白作用于FG,在12 h内即可观察到细胞的死亡;酶对大菱鲆也表现出致死毒性,其症状与感染鳗弧菌的病鱼症状相似,半致死剂量LD50为8.1μg/鱼。
利用PCR方法对推测的EmpA金属蛋白酶活性位点氨基酸的碱基序列进行了定点突变,将突变质粒转化到大肠杆菌BL21(DE3)中进行表达,并用Ni亲合柱分别纯化了各突变蛋白。比较EmpA金属蛋白酶及其突变蛋白的蛋白水解活性和细胞毒性发现,突变蛋白都不同程度地丧失了其蛋白水解活性和细胞毒性,表明突变位点的氨基酸对酶活性起着十分重要的作用。
将没有蛋白水解活性和细胞毒性的m-EmpA7蛋白的基因序列m-empA7克隆到真核表达载体pEGFP-N1中,得到真核重组表达质粒pEGFP-N1/m-empA7。用磷酸钙共沉淀法将重组质粒转染至CHO和HEK293T细胞中,通过荧光显微镜、RT-PCR检测和Western-blot检测证实目的基因在真核细胞内获得了表达。
对牙鲆肌肉注射50μg/尾的重组质粒pEGFP-N1/m-empA7,于接种后不同时间分别取对照组和试验组牙鲆各组织做冷冻切片,在荧光显微镜下观察可见,在试验组牙鲆的注射点周边肌肉、注射点对面体侧肌肉、脾脏、前肾、体肾、后肠、心、鳃和肝脏等部位均可见绿色荧光,对照组组织没有荧光。RT-PCR检测和Western-blot检测的结果也证实目的基因在牙鲆体内获得了表达。
为研究真核重组表达质粒pEGFP-N1/m-empA7对牙鲆的免疫效果,分别以5μg/尾、20μg/尾和50μg/尾的不同剂量对牙鲆进行肌肉注射免疫,同时设置注射PBS和空质粒的对照组。ELISA检测结果显示,各免疫组牙鲆体内均产生了抗m-EmpA7抗原的特异性抗体,且抗体水平随接种剂量的增加而升高;而对照组牙鲆血清基本没有抗体凝集发生,在不同时间内抗体水平无明显变化。免疫后第5周,用鳗弧菌W-1对各组进行攻毒实验,各免疫组的免疫保护率(RPS)分别为57.1%,71.4%和85.7%,与对照组相比,免疫组产生了显著的保护作用(p < 0.001)。
Vibrio anguillarum is the causative agent of vibrosis, one of the major bacterial disease affecting marine and freshwater fishes in the world. The infected fish showed a rapid fulminating hemorrhagic septicemia and wide-spread tissue damage, and died in several days. The distribution of vibrosis is worldwide, which cause great economic loss to the aquaculture industry. Vibrio anguillarum strain W-1 was originally isolated from diseased seabass (Lateolabrax japonicus). Previous studies revealed that the extracellular protease EmpA of Vibrio anguillarum was one its important virulence factors. The gene coding EmpA was cloned and sequenced.
In this study, empA was cloned into three prokaryotic expression plasmids pBV220, pBAD24 and pET24d(+) respectively, resulting three recombinant Escherichia coli strains JM109/pBV220/empA、TOP10/pBAD24/empA and BL21(DE3)/pET24d(+)/empA. We finally chose BL21(DE3)/pET24d(+)/empA to express, purify and mutate empA since comparisons of the expression of empA in this three recombinant strains showed that BL21(DE3)/pET24d(+)/empA had the best expression profile. The expression conditions of the recombinant E. coli BL21(DE3)/pET24d(+)/empA were optimized. It was found that the soluble productions of EmpA expressed in recombinant cells and secreted to the medium were both maximal when E. coli BL21(DE3)/pET24d(+)/empA was induced with 1 mM IPTG at 25℃. The expressed reombinant EmpA could react with the anti-EmpA serum in Western-blot. EmpAs expressed in recombinant E. coli and secreted to the medium were purified by metal chelating affinity chromatography. Estimated molecular weights of purified EmpAs were both 36 kDa by SDS-PAGE, but both 44.6 kDa if the samples were not heated in boiling water. And they were both determined to be 44.6 kDa by Sephadex G-200 gel filtration. Thus, EmpA expressed in E. coli was a monomer polypeptide. Purified EmpA had cytotoxicity to flounder gill cell line (FG). The development of CPE-like morphologic damage of the cells was visible by light microscopy within 12 h of incubation of the cells with 5μg of purified EmpA. It could also cause death of turbot when injected intraperitoneally, which showed similar symptoms with diseased fish infected by Vibrio anguillarum. The LD50 of EmpA to turbot was established as 8.1μg protein/g of fish.
The nucleotides of the amino acid residues in the active sites of EmpA were mutated by PCR site-directed mutagenesis. All recombinant plasmids with mutated empAs were transformed into E. coli BL21(DE3), and mutated EmpAs were expressed and purified to characterize the effects of alterations of these amino acid residues on enzymatic activity. It was found that all of the single point mutations at the conservative residues reduced the proteolytic activity and cytotoxicity of the enzyme with different levels, indicating that they play an important role in enzymatic activity.
To construct DNA vaccine, mutated empA (m-empA7) was subcloned into eukaryotic expression vector pEGFP-N1, resulting the recombinant plasmid pEGFP-N1/m-empA7. It was then transfected into CHO and HEK293T cells and the expression of m-empA7 was detected with fluorescent microscope, RT-PCR and Western-blot, which indicating that m-empA7 was expressed in CHO and HEK293T cells.
Flounder were intramuscularly injected with 50μg of the DNA vaccine solution to examine if m-empA7 could be expressed. Fluorescent microscope, RT-PCR and Western-blot detection revealed that m-empA7 was expressed in the muscle, spleen, kidney, hind intestines, heart, gill and liver of flounder.
To study the protective efficacy of the recombinant plasmid pEGFP-N1/m-empA7 on flounder, fish were respectively immunized with three doses of DNA vaccine (5μg/fish, 20μg /fish and 50μg /fish). The results of ELISA revealed that the immunized fish harvested much higher special antibody against m-EmpA7 in relation to negative controls of PBS buffer and pEGFP-N1, and the antibody titer increased as the immunized dose increased. Five weeks after vaccination, fish were challenged by intramuscularly injection of Vibrio anguillarum W-1 cell suspension. Mortalities following exposure to the bacteria were much lower in fish vaccinated with pEGFP-N1/m-empA7 compared to those of the control groups injected with PBS buffer and pEGFP-N1 alone. DNA vaccine of three doses protected fish from the bacterium with 57.1%, 71.4% and 85.7% of RPS respectively.
引文
陈吉祥,刘霜,李筠,王祥红,杜宗军,于德华,纪伟尚,徐怀恕。一种鳗弧菌胞外蛋白酶的分离纯化及性质。中国水产科学,2002a,4:318-322。
陈吉祥,李筠,王祥红,杜宗军,于德华,纪伟尚,徐怀恕。致病性鳗弧菌金属蛋白酶基因克隆及序列分析。高技术通讯,2002b,6:106-110。
邓国成,姜兰。加州鲈鱼(Micropterus salmoides)细菌性烂尾、烂嘴病病原的研究。中国水产科学,1996,3(4):83-92。
方维焕,梁雪芽。减毒沙门氏菌为载体在Vero细胞中表达新城疫病毒融合蛋白。生物化学与生物物理学报,2002,34(4):488-493。
黄琪琰。中国水产动物疾病学研究进展。水产学报,1996,20(1):51-57。
李军,冯娟,徐怀恕。香港地区养殖平鲷的病原菌(溶藻胶弧菌)研究。水产学报,1982,22(3):275-278。
李清禄,陈强。海水网箱养殖大黄鱼细菌性病原鉴定与感染治疗研究。应用与环境生物学报,2001,7:489-493。
彭贵洪,马忠,薛宇鸣。尿激酶变体Glu154-mtcu-PA的构建及其性质研究。生物化学与生物物理学报,1997,29(6):547-552。
覃映雪,苏永全,周化民。副溶血弧菌(Vibrio parahaemolyticus)极鞭毛蛋白FlaB基因的克隆与序列分析。农业生物技术学报,2003,11(6):656-657。
史瑾,魏家绵,沈允钢。玉米叶绿体ATP合成酶的定点突变。生物化学与生物物理学报,1998,30(5):483-487。
王保坤,余俊红,李筠,纪伟尚,徐怀恕。花鲈弧菌病原菌(哈维氏弧菌)的分离与鉴定。中国水产科学,,2002,1:52-55。
肖慧,李军,王祥红,纪伟尚,徐怀恕。鲈鱼苗烂鳃、烂尾病病原菌的研究。青岛海洋大学学报(自然科学版),1999,29:87-93。
徐怀恕,杨学宋,李筠。对虾细菌苗期细菌病害的诊断于控制。北京:海洋出版社,1999。
孙修勤,郑风荣,李继业,吴兴安,曲凌云,张进兴,洪旭光。牙鲆淋巴囊肿病毒主要衣壳蛋白基因片段真核表达载体的构建、表达与免疫效果评价。高技术通讯,2006,16(7):740-745。
谢海侠,聂品,王桂堂。鱼用核酸疫苗研究进展。水生生物学报,2004,28(1):90-94。
郑国兴,沈亚林,李何。中国对虾病原菌(鳗弧菌)的研究。水产学报,1990,14(1):1-7。
焉庆批,苏永全,王军。绿色荧光蛋白基因在大黄鱼体内的表达。厦门大学学报(自然科学版),2002,41(2):265-268。
周化民,王军,苏永全。含副溶血弧菌(Vibrio parahaemolyticus)FlaⅠ基因真核表达重组质粒的构建。厦门大学学报(自然科学版),2002,42(40):21-27。
Actis LA, Tolmasky ME, Farrell DH and Crosa JH. Genetic and molecular characterization of essential components of the Vibrio anguillarum plasmid-mediated iron-transport system. J Biol Chem, 1988, 263: 2853-2860.
Aeckersberg F, Lupp C, Feliciano B. Vibrio fischeri outer membrane protein ompU plays a role in normal symbiotic colonization. J Bacteriol, 2001, 183(220): 6590-6597.
Ahne W, Popp W and Hoffman R. Pseudomonas fluoresens as a Pathogen of tencha(Tinca tinca). Bulletin of the European Association of Fish Pathologists, 1982, 4: 56-57.
Amandi A, Hiu SF, Rohovec JS. Isolation and characterization of Edwardsiella tarda from Chinook Salmon (Oncorhynchus tshawyscha). Appl Environ Microbiol, 1982, 43: 1380-1384.
Anderson ED, Mourich DV, Fahrenkrug SC. Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Mol Marine Biol Biotechnol, 2000, 5: 114-122.
Austin B, Austin DA. Bacterial fish pathogens: diseases in farmed and wild fish.London, United Kingdom: Springer, 1999, 3rd edn.
Bernoth EM, Ellis AE, Midtly PJ. Furunculosis Multidisciplinary Fish Disease Research. San Dieqo, Academic Press: 1997.
Barry MA, Lai CL, Johnston SA. Protection against myooplasma infection using expression-library immunization. Science, 1996, 377: 632-635.
Benjamin ES, Jo-Ann C. Gene Transfer to Fish Cells by Attenuated Invasive Escherichia coli. Biotechnol, 2002, 4: 303-309.
Boesen HT, Pedersen K, Larsen JL, Koch C, Ellis AE. Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide. Infect Immun, 1999, 67(1): 293-301.
Boesen HT, Pedersen K, Larsen JL. Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss).serum: role of O-antigen structure of lipoploysaccharide. Infect Immun, 1999, 67(1): 293-301.
Booth BA, Finkelstein MB, Finkelstein RA. Vibrio cholerae soluble hemagglutinin/protease is a metalloenzyme . Infect Immun, 1984, 42: 639-644.
Booth A, Boesman-Finkelsrein M, Finkelsrein RA. Vibrio cholerae hemagglutinin/protease nicks cholera enterotoxin. Infect Immun,1984, 45: 558-560.
Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of proteins using the principle of protein-dye binding. Anal Biochem, 1976, 72: 248-54.
Carl T, Makoto E, Ikuo H. Assessment of DNA vaccine potential for juvenile japanese flounder Paralichthy-solivaceus, through the introduction of reporter genes by particle bombardment and histopathology. Vaccine, 2001, 19: 801-809.
Chart H. Multiflagellate variants of Vibrio anguillarum. J Gen Microbiol, 1983, 129: 2193-2197.
Chart H and Trust TJ. Characterization of the surface antigens of the marine fishPathogens, Vibrio anguillarum and Vibrio ordalii. Can Journal Microbiol, 1984, 30: 703-710.
Chen FR, Liu PC, Leek K. An evaluation of chromogenic substrates for characterization of serine protease produced by pathogenic Vibrio alginolyticus. Microbiol, 1999, 98(3): 27-34.
Chen JX, Li Y, Gou WL. An extracellular protease of Vibrio anguillarum: preparation of the enzyme, its physicochemical properties, gene clone and potential use in DNA vaccine. Postgraduate Conference on Marine Biology and Biotechnology, Hong Kong: 6-8 June 2001.
Chinose Y, Ehara M, Honda T. The effect on enterotoxicity of protease purified from Vibrio cholerae Ol. FEMS Microbiol Lett, 1994, 115: 265-271.
Colwell RR and Griems DJ. Vibrio diseases of marine fish populations. Helgoland. Meeresunters, 1984, 37: 265-287.
Chowdhury MAR, Miyoshi SI, Shinoda S. Vascular permeability enhacement by Vibrio mimicus protease and the mechanisms of action. Microbiol Immunol, 1991, 35: 1049-1058.
Crosa JH. A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an ironsequestering system. Nature, 1980, 284: 566-568.
Cornelis GR, Van Gijsegem F. Assembly and function of type III secretory systems. Annu Rev Micriobiol, 2000, 54: 735-74.
Dai JL, Bansal RK, Kern SE. G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation. Proc Natl Acad Sci: USA, 1999, 96 (4): 1427-1432.
Davey ML, Hancock REW, Mutharia LM. Influence of culture conditions on exprssion of the 40 kilodolton porin protein of Vibrio anguillarum serotype O2. Appl Environ Microbiol, 1998, 64: 138-246.
David VA, Deutch AH, Sloma A, Pawlyk D, Aly A, Durham DR. Cloning, sequencing and expression of the gene encoding the extracellularneutral protease, vibriolysin, of Vibrio proteolyticus. Gene, 1992, 112: 107-112.
Davis HL. Development of DNA vaccines for fish: vector design, intramuscular injection and antigene expression using viral haemorrhagic septicemia virus genes as mode. Fish Shellfish Immunol, 1998, 8: 271-286.
Denkin SM, Nelson DR. Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence. Appl Environ Microbiol, 2004, 70: 4193-4204.
Dickerson H, Clark T, Lin TL. Diagnostic and protective antigen gene sequences of Ichthyophirius multifiliis. USA: M & R 235, 00170201, 2000-02-16.
Egidius E. Vibriosis: pathogenicity and pathology, a review. Aquaculture, 1987, 67: 15-18.
Eldar A, Bejerano Y and Bercover H. Streptococcus shiloi and Streptococcus difficile: two new Streptococcal Species causing a meningoecephalitis in fish. Curr Microbiol, 1994, 28: 139-143.
Farrell DH, Crosa JH. Purification and characterization of a secreted protease from the pathogenic marine bacteria Vibrio anguillarum. Biochem, 1991, 30: 3422-3436.
Fletcher MC, Kuderova A, Cygler M. Creation of a ribonuclease abzyme through site-directed mutagenesis. Nat Bio technol, 1998, 16(11): 1065-1067.
Franco AA, Buckwold SL, Shin JW, Ascon M and Sears CL. Mutation of the Zinc-Binding Metalloprotease Motif Affects Bacteroides fragilis Toxin Activity but Does Not Affect Propeptide Processing. Infect Immun, 2005, 73: 5273-5277.
Fukasawa S, Nakamura K, Kamii A. Purification and properties of a protease from a marine luminous bacterium, Vibrio harveyi strain FLA-ll. Agri Biol Chem, 1988, 52: 435-441.
Hase CC, Finkelstein RA. Cloning and nucleotide sequence of the Vibriocholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain. J Bacteriol, 1991, 173: 3311-3317.
Helen LB, Kate FG, Steven MJ. Antibody response to yersinia pestis flantigen expressed in Salmonella typhimurium aroA from in vivo-inducible promoters. Vaccine, 2000, 18: 2668-2676.
Hensen E, Fernandez K, Goldspink G, Butterworth P, Umeda PK, Chang KC. Strong expression of foreign genes following direct injection into fish muscle. FEBS Lett, 1991, 1(290): 73-76.
Heppell J, Lorenzen N, Armstrong NK, Wu T, Lorenzen E, Einer-jensen K, Schorr J, Davis HL. Development of DNA vaccines for fish: vector design, intramuscular injectin and antigenexpression using viral haemorrhagic septicemia virus genes as model. Fish Shellfish Immonol, 1998, 8: 271-286.
Hirono I, Masuda T and Aoki T. Cloning and detection of the hemolysin gene of Vibrio anguillarum. Microbiol Pathoqenesis, 1996, 21: 173-182.
Horne MT and Baxende A. The adhesion of Vibrio anguillarum to host tissues and its roles in pathogenesis. J Fish Diseae, 1983, 6: 461-471.
Ichinose Y, Yamamoto K, Nakasome N. Enterotoxicity of El Tor-like hemolysin of non-Ol Vibrio cholerae. Infect Immun, 1987, 55: 1090-1093.
Ichinose Y, Ehara M, Honda T. The effect on enterotoxicity of protease purified from Vibrio cholerae Ol. FEMS Microbiol Lett, 1994, 115: 265-271.
Inamura H, Nakai T, Muroga K. An extracellular protease produced by Vibrio anguillarum. Bull Jpn Soc Sci Fish, 1985, 51: 1915-1920.
Ingunn S, Lorenzen E, Lorenzen N. A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus in turbot. Vaccine, 2003, 21: 4661-4667.
Joel H, Heather LD. Application of DNA vaccine technology to aquaculture. Adv Drug Deliver Rev, 2000, 43: 29-43.
Jorgensen JB, Johansen A, Stenersen B. CpG oligodeoxynucleotides and plasmid DNA simulate Atlantic salmon (salmo salar L.) leucocytes toproduce supernatants with antiviral activity. Dev Comp Immunol, 2001a, 25: 313-21.
Jorgensen JB, Zhou J, Johansen A. Immunostimulatory CpG oligodeoxynucleotides simulate expression of IL-1βand interferon-like cytokines in rainbow trout macrophages via a chloroquine-sensitive mechanism. Fish Shellfish Immunol, 2001b, 11: 673-82.
Kammann M, Laufs J, Schell J. Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acid Res, 1989, 17(13): 5404.
Kanemori Y, Nakai T. and Muroga K. The role of Extracellular protease produced by Vibrio anguillarum. Fish Pathology, 1987, 22(3): 153-158.
Kanno T, Nakai T and Muroga K. Scanning electron microscopy on the skin surface of ayu plecoglossus altivelis infected with Vibrio anguillarum. Dis Aquat Organ, 1990, 8: 73-75.
Karan MG, Horstedt P, Milton DL. Identification and characterisation of additional flagellin genes from Vibrio anguillarum. J Bacteriol, 1996, 178(9): 5188-5198.
Kawamoto S, Shibano Y, Fukushima J, Ishii N, Morihara K and Okuda K. Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection. Infect Immun, 1993, 61: 1400-1405.
Kim SK, Yang JY, Cha J. Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04. Gene, 2002, 283: 277-286.
Kono M, Miyaza KIG, Nakamu RAH. Site-directed mutagenesis in hemoglobin: at tempts to control the oxygen affinity with cooperativity preserved.Protein Eng, 1998, 11(3): 199-204.
Kooi C, Corbett CR and Sokol PA. Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease. J Bacteriol, 2005, 187: 4421-4429.
Kothary MH, Kreger AS. Purification and characterization of an elastolytic protease of Vibrio vulnificus. J Gen Microbiol, 1987, 133: 1783-1791.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 277: 680-685.
Laird AD, Morrison DK, Shalloway D. Characterization of Raf-1 activation in mitosis. J Biol Chem, 1999, 274 (7): 4430-4439.
Laygren B, Fausa PE, Wergeland H. An extracellular low molecular weight protease with activity against gelatin and casein produced earlier during growth of Aeromonas salmonicida ssp,Salmonicida than the serine protease. Bull Euro Assoc Fish pathol, 1998, 18: 96-101.
Lee CY, Su SC, Liaw RB. Molecular analysis of an extracellular protease gene from Vibrio parahaemolyticus.Microbiol, 1995, 141: 2569-2576.
Lee JH, Kim GT, Lee TY. Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus. BBA, 1998, 1384: 1-6.
Lee CY, Cheng MF, Yu MS. Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus. FEMS Microbiol Lett, 2002, 209(1): 30-36.
Lida T and Honda T. Hemolysins produced by Vibrios.Journal of Toxicology Toxin Rev, 1997, 16: 215-227.
Lorenzen N, Lorenzen E, Einer-Jensen K. DNA vaccines as a tool for analyzing the protective immune response against rhabdoviruses in rainbow trout. Fish Shellfish Immunol, 2002a, 12: 439-453.
Lorenzen N, Lorenzen E, Katja E J. Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens. Developmental and comparative Immunology, 2002b, 26: 173-179.
Lorenzen N, Niels JO, Claus K. Immunity to VHS virus in rainbow trout. Aquaculture, 1999, 172: 41-61.
Lorenzen N, Lorenzen E, Katja EJ. Immunity to viral haemorrhagic septicaemia (VHS) following DNA vaccination of rainbow trout at an early life-stage. Fish & Shellfish Immunology, 2001, 11: 585-591.
Kanellos TS, Sylvester ID. Mammalian granulocytemacrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish. Immunology, 1999, 96: 507-510.
Kristine R, Beate JT, Oystein E. Expression of luciferase in selected organs following delivery of naked and formulated DNA to rain bow trou t (Oncorhynchus mykiss) by different routes of administration. Fish Shellfish Immunol, 2004, 16: 251-264.
Kumar SR, Parameswaran V, Ishaq Ahmed VP, Musthaq SS, Sahul Hameed AS. Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum. Fish Shellfish Immunol, 2007 Aug, 23(2): 316-26.
Maeda H. Role of microbial protease in pathogenesis. Microbiol Immunol, 1996, 40: 685-699.
Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selectior. Proc Natl Acad Sci, USA: 1985, 82 (2): 488-492.
Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Micriobiol Rev, 1996, 60: 512-538.
Mergulhao FJM, Monteiro GA, Cabral JMS, Taipa MA. Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. J Microbiol Biotechnol, 2004, 14: 1-14.
Marta A, Marc J, Ben S. A fish specific expression vector containing the interferon regulatory factor 1A (IRF1A) promoter for genetic immunization of fish. Vaccine, 2003, 21: 1591-1600.
Maureen KP, Gael K, Kyle AG. Quantitative expression profiling of immuneresponse genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) Infection or DNA vaccination. Fish & Shellfish Immunology, 2004, 17: 447-462.
McIver K, Kessler E and Ohman DE. Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase. J Bacteriol, 1991, 173: 7781-7789.
McIver KS, Olson JC and Ohman DE. Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion. J Bacteriol, 1991, 175: 4008-4015.
Michael JM, Heather LD. CpG DNA as mucosal adjuvant. Vaccine, 2000, 18: 231-237.
Mikalsen AB, Sindre H, Torgersen J, Rimstad E. Protective effects of a DNA vaccine expressing the infectious salmon anemia virus hemagglutinin-esterase in Atlantic salmon. Vaccine, 2005, 23: 4895-4905.
Milton DL, Norqvist A, Wolf-Watz H. Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum. J Bacteriol, 1992, 174: 7235-7244.
Milton DL, Norqvist A, Wolf-Watxtz H. Sequence of a novel virulence-mediating gene, virC, from Vibrio anguillarum. Gene, 1995, 164(1): 95-100.
Milton DL, O' Toole R, Horstedt P, Wolf-Watz H. Flagellin A is essential for the virulence of Vibrio anguillarum J Baoteriol, 1996, 178(5): 1310-1319.
Miyoshi S, Sugiyama K, Suzuki Y. Enhancement of vascular permeability due to histamine-releasing effect of Vibrio vulnificus protease. FEMS Microbiol Lett, 1987, 40: 95-98.
Miyoshi S, Wakae H, Tomochika. Functional domains of a zinc metalloprotease from Vibrio vulnificus. J Bacteriol, 1997, 179: 7606-7610.
Miyoahi S, Nakazawa H, Tomochika K. Characterization of the hemmorrhagic reaction caused by Vibrio vulnificus metallopretease, a member of the thermolysin family. Infect Immun, 1998, 66: 4851-4855.
Miyoshi DL, Nakazawa H, Kawata K. Characterization of the hemorrhagic reaction caused by Vibrio vulnificus metalloprotease, a member of the hemolysin family. Infect Immune, 1998, 66: 4851-4855.
Mo ZL, Tan XG, Xu YL. A Vibrio anguillarum strain associated with ulcerative skin in cultured flounder, Paralichthys olivaceus. Chin J Oceano Lumino, 2001, l19(4): 319-326.
Montero AB and Austin B. Characterization of extracellular products from an isolate of Vibrio harveyi recovered from diseased post-larval Penatus vannamei (Bonne). J Fish Dis, 1999, 22: 377-386.
Mukhopadhya YP, Roy KB. Protein engineering of BamHI restriction endonuclease: replacement of Cys54 by Ala enhances catalytic activity. Protein Eng, 1998, 11(10): 931-935.
Munn CB. Haemolysin production by Vibrio anguillarum. FEMS Microbiol Lett, 1978, 3: 265-268.
Mun CB. Production and properties of a haemolytic toxin by Vibrio anguillarum. Fish Diseases. Third copraq session, Springer-Verlag, Berlin: 1980, 69-74.
Muroga K. Viral and bacterial diseases of marine fish and shellfish in Japanese hatcheries. Aquaculture, 2001, 202: 23-24.
Nakamura Y, Nakai T, Muroga K. Induction of antibody producing cells in some fishes by immunization with Vibrio anguillarum O-antigen. Fish Pathol, 1990, 25: 225-230.
Nieto TP and Ellis AE. Characterization of extracellular metalo-and serine-protease of Aeromonas hydrophila strain B51. Journal of Gen Microbiol, 1986, 132: 1975-1979.
Nishibuchi M and Kaper JB. Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus. J Bacteriol, 1985, 162:558-564.
Nishibuchi M, Khaeomaneeiam V, Honda T. Comparative analysis of the hemolysin qenes of Vibrio cholerae non-Ol, V. mimicus,and V. hollisae that are similar to the tdh gene of V. parahaemolyticus. FEMS Microbiol Lett, 1990, 67: 251-256.
Noonan B, Enzmann PJ, Trust TJ. Recombinant infectious hematopoietic nercosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromanas salmonicida induce protective immunity in rain trout (Oncorhynchus mykiss). Appl Environ Microbiol, 1995, 61(10): 3586-3591.
Norqvist A, Norrman B, Wolf-Watz H. Identification and characterization of a zinc metalloprotease associated with invasion by the fish pathogen Vibrio anguillarum. Infect Immun, 1990, 58: 3731-3736.
Norqvist A, Wolf-Watxtz H. Characterization of a novel chromosomal virulence locus involved in expression of a major surface flagellar sheath antigen of the fish pathogen Vibrio anguillarum. Infect Immun, 1993, 61(6): 2434-2444.
Olsson JC, Joborn A, Westerdahl A. Is the turbot, Scophthalmus maximus(L), intestine a partial of entry for the fish pathogen Vibrio anguillarum. J Fish Disease, 1996, 19: 225-234.
Ormonde P, Hoerstedt P, O’Toole R. Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum. J Bacteriol, 2000, 182(8): 2326-2328.
Pathiratne A, Widauapathiraua G.S and Chandrosrkanthi WHS. Association of Aeromonas hydrophila with epizootic ulcerative syndrome (EUS) in freshwater fish in Sri Lanka. Journal Appl Ichthyol, 1994, 10: 204-208.
Pavlovskis OR, Wretlind B. Assessment of protease (elastase)as a Pseudomonas aeruginosa virulence factor in experimental mouse burn infection. Infect Immun, 1979, 24: 181-187.
Pederson K, Gram L, Austin DA. Pathogenicity of Vibrio anguillarum serogroup Ol compared to plasmids,outer membrane protein profile,and siderophore production. J Appl Microbiol, 1997, 82: 365-371.
Pyle SW and Shotts EB. A new approach for differiating flexihacferia isolated from cold-water and warm-water fish. Can J Fish Aquat Sci, 1980, 37: 1040-1042.
Ransom DP. Bacteriologic immunologic and pathologic studies of Vibrio sp. pathogenic to salmonids. Ph.D.thesis, Oregen State University, Corvallis: 1978.
Rawlings ND and Barrett AJ. Evolutionary families of metallopeptidases. Methods Enzymol, 1995, 248: 183-228.
Salati F, Watanabe K, Kawai K et al. Immune response of ayu against Vibrio anguillarum Lipopolysaccharide, Nippon Suisan Gakkaishi,1989,55:45-49
Seo JY, Kim KH, Kim SG, Oh MJ, Nam SW, Kim YT, Cho TJ. Protection of flounder against hirame rhabdovirus (HIRRV) with a DNA vaccine containing the glycoprotein gene. Vaccine, 2006, 24: 1009-1015.
Shinoda S. Protein toxins produced by pathogenic vibrios. Journal of Natural Toxicon, 1999, 8: 259-269.
Simon M, Mathes A, Blanch A. Characterization of a porin from the outer membrane of Vibrio anguillarum. J Bacteriol, 1996, 178: 4182-4188.
Singer J T, Schmidt K A, and Reno P W. Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virolence of the marine fish pathogen Vibrio anguillarum. Bacteriology, 1991, 173: 1347-1352.
Suzuki S, Kuroe K, Yasue K. Antigenicity and N-terminal amino acid sequence of a 35 kDa porin-like protein of Listonella (vibrio) anguillarum: comparison among different serotypes and other bacterial species. Lett Appl Microbiol, 1996, 23(5): 303-306.
Takeuchi H, Shibano Y, Morihara. Structural gene and complete amino acidsequence of Vibrio alginolyticus collagenase. Biochem J, 1992, 281: 703-708.
Terai A, Shirai H, Yoshida O. Nucleotide sequence of the thermostable direct hemolysin qene(tdh gene)of Vibrio mimicus and its evolutionary relationship with the tdh genes of Vibrio Parahaemolyticus. FEMS Microbiol Lett, 1990, 71: 319-324.
Thune RL, Graham TE, Riddle LM. Extracellular proteases from Aeromonas hydrophila. partial purification and effects on aqeochannel catfish. Transact Am Fish soc, 1982, 111: 749-754.
Tiziano V, Laura I, Rita C. Assessment of DNA vaccine potential for gilthead sea bream (Sparus aurata) by intramuscular injection of a reporter gene. Fish Shellfish Immunol, 2003, 15: 283-295.
Tolmaskg ME and Crosa JH. Iron transport genes of the pJMl-mediated iron uptake system of Vibrio anguillarum are included in a transposon like strcture. Plasmid, 1995, 37: 180-190.
Tomokazu T, Akiko I, Ikuo H. Development of a DNA vaccine against hiramerhabdovirus and analysis of the expression of immune-related genes after vaccination. Fish & Shellfish Immunology, 2004, 17: 367-374.
Tsunasawa S, Suginara A, Masaki T. Amino acid sequence of termostable direct hemolysin produced by Vibrio Parahaemolyticus. J Bioch, 1987, 101: 111-121.
Tuomanen El. Cell adhesion molecules in the development of bacterial infections in leukocyte adhesion basic and clinical aspects. (Gamberg CG, Mandrap-PoulsenT and Wogensenbach L eds), Elsevier Sciencc Publishers, New York: 1992, 297-306.
Ulmer JB, Donnely J J, Parker SE. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 1993, 59: 1745-1749.
von Heijne G. A new method for predicting signal sequence cleavage sites.Nucleic Acids Res, 1986, 14: 4683-4690.
Wane A, Pakl KA. Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability. Biochem, 1998, 37(38) : 13120- 13127.
Wang XH, Oon HGW. Internalization and cytotoxicity are important virulence mechanisms in vibrio-fish epithelial cell interactions. Micro, 1998, 144(11): 2987-3002.
Wolff IA, Malone RW, Williams P. Genetic immunization is a simple method for eliciting an immune respones. Nature, 1992, 356: 152-154.
Wolke RE. Pathology of bacteriol and fungal disease affecting fish In:Ribelin W E and Migaki G(eds), The Pathology of Fishes. Wisconsin: University of Wisconsin Press, 1975: 76-78.
Yamamoto K, Ichinose Y, Shinagawa H. Two-step processing for activation of the cytolysin/ hemolysin of the structural gene (hlyA) and characterization of the processed products. Infect Immun, 1990, 58: 4106-4116.
Yamasoki S, Shirai H, Takeda Y. Analysis of the qene of Vibrio hollisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus. FEMS Microbiol Lett, 1991, 80: 259-264.
Yi AK, Tuetken R, Redford T. CpG motifs in bacterial DNA activate oxygenspecied. Immunol, 1998, 160: 4755-4761.
Zhen M, Jianzhong S, Lixin X. CpG oligodeoxynucleotides activate grass carp (ctenopharyngo don idellus) macrophages. Dev Com Immunol, 2003, 27: 313-321.
Zheng FR, Sun XQ, Liu HX, Zhang JX. Study on the distribution and expression of a DNA vaccine against lymphocystis disease virus in Japanese flounder(Paralichthys olivaceus). Aquaculture, 2006, 261: 1128-1134.
陈吉祥,李筠,王祥红,杜宗军,于德华,纪伟尚,徐怀恕。致病性鳗弧菌金属蛋白酶基因克隆及序列分析。高技术通讯,2002b,6:106-110。
邓国成,姜兰。加州鲈鱼(Micropterus salmoides)细菌性烂尾、烂嘴病病原的研究。中国水产科学,1996,3(4):83-92。
方维焕,梁雪芽。减毒沙门氏菌为载体在Vero细胞中表达新城疫病毒融合蛋白。生物化学与生物物理学报,2002,34(4):488-493。
黄琪琰。中国水产动物疾病学研究进展。水产学报,1996,20(1):51-57。
李军,冯娟,徐怀恕。香港地区养殖平鲷的病原菌(溶藻胶弧菌)研究。水产学报,1982,22(3):275-278。
李清禄,陈强。海水网箱养殖大黄鱼细菌性病原鉴定与感染治疗研究。应用与环境生物学报,2001,7:489-493。
彭贵洪,马忠,薛宇鸣。尿激酶变体Glu154-mtcu-PA的构建及其性质研究。生物化学与生物物理学报,1997,29(6):547-552。
覃映雪,苏永全,周化民。副溶血弧菌(Vibrio parahaemolyticus)极鞭毛蛋白FlaB基因的克隆与序列分析。农业生物技术学报,2003,11(6):656-657。
史瑾,魏家绵,沈允钢。玉米叶绿体ATP合成酶的定点突变。生物化学与生物物理学报,1998,30(5):483-487。
王保坤,余俊红,李筠,纪伟尚,徐怀恕。花鲈弧菌病原菌(哈维氏弧菌)的分离与鉴定。中国水产科学,,2002,1:52-55。
肖慧,李军,王祥红,纪伟尚,徐怀恕。鲈鱼苗烂鳃、烂尾病病原菌的研究。青岛海洋大学学报(自然科学版),1999,29:87-93。
徐怀恕,杨学宋,李筠。对虾细菌苗期细菌病害的诊断于控制。北京:海洋出版社,1999。
孙修勤,郑风荣,李继业,吴兴安,曲凌云,张进兴,洪旭光。牙鲆淋巴囊肿病毒主要衣壳蛋白基因片段真核表达载体的构建、表达与免疫效果评价。高技术通讯,2006,16(7):740-745。
谢海侠,聂品,王桂堂。鱼用核酸疫苗研究进展。水生生物学报,2004,28(1):90-94。
郑国兴,沈亚林,李何。中国对虾病原菌(鳗弧菌)的研究。水产学报,1990,14(1):1-7。
焉庆批,苏永全,王军。绿色荧光蛋白基因在大黄鱼体内的表达。厦门大学学报(自然科学版),2002,41(2):265-268。
周化民,王军,苏永全。含副溶血弧菌(Vibrio parahaemolyticus)FlaⅠ基因真核表达重组质粒的构建。厦门大学学报(自然科学版),2002,42(40):21-27。
Actis LA, Tolmasky ME, Farrell DH and Crosa JH. Genetic and molecular characterization of essential components of the Vibrio anguillarum plasmid-mediated iron-transport system. J Biol Chem, 1988, 263: 2853-2860.
Aeckersberg F, Lupp C, Feliciano B. Vibrio fischeri outer membrane protein ompU plays a role in normal symbiotic colonization. J Bacteriol, 2001, 183(220): 6590-6597.
Ahne W, Popp W and Hoffman R. Pseudomonas fluoresens as a Pathogen of tencha(Tinca tinca). Bulletin of the European Association of Fish Pathologists, 1982, 4: 56-57.
Amandi A, Hiu SF, Rohovec JS. Isolation and characterization of Edwardsiella tarda from Chinook Salmon (Oncorhynchus tshawyscha). Appl Environ Microbiol, 1982, 43: 1380-1384.
Anderson ED, Mourich DV, Fahrenkrug SC. Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Mol Marine Biol Biotechnol, 2000, 5: 114-122.
Austin B, Austin DA. Bacterial fish pathogens: diseases in farmed and wild fish.London, United Kingdom: Springer, 1999, 3rd edn.
Bernoth EM, Ellis AE, Midtly PJ. Furunculosis Multidisciplinary Fish Disease Research. San Dieqo, Academic Press: 1997.
Barry MA, Lai CL, Johnston SA. Protection against myooplasma infection using expression-library immunization. Science, 1996, 377: 632-635.
Benjamin ES, Jo-Ann C. Gene Transfer to Fish Cells by Attenuated Invasive Escherichia coli. Biotechnol, 2002, 4: 303-309.
Boesen HT, Pedersen K, Larsen JL, Koch C, Ellis AE. Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide. Infect Immun, 1999, 67(1): 293-301.
Boesen HT, Pedersen K, Larsen JL. Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss).serum: role of O-antigen structure of lipoploysaccharide. Infect Immun, 1999, 67(1): 293-301.
Booth BA, Finkelstein MB, Finkelstein RA. Vibrio cholerae soluble hemagglutinin/protease is a metalloenzyme . Infect Immun, 1984, 42: 639-644.
Booth A, Boesman-Finkelsrein M, Finkelsrein RA. Vibrio cholerae hemagglutinin/protease nicks cholera enterotoxin. Infect Immun,1984, 45: 558-560.
Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of proteins using the principle of protein-dye binding. Anal Biochem, 1976, 72: 248-54.
Carl T, Makoto E, Ikuo H. Assessment of DNA vaccine potential for juvenile japanese flounder Paralichthy-solivaceus, through the introduction of reporter genes by particle bombardment and histopathology. Vaccine, 2001, 19: 801-809.
Chart H. Multiflagellate variants of Vibrio anguillarum. J Gen Microbiol, 1983, 129: 2193-2197.
Chart H and Trust TJ. Characterization of the surface antigens of the marine fishPathogens, Vibrio anguillarum and Vibrio ordalii. Can Journal Microbiol, 1984, 30: 703-710.
Chen FR, Liu PC, Leek K. An evaluation of chromogenic substrates for characterization of serine protease produced by pathogenic Vibrio alginolyticus. Microbiol, 1999, 98(3): 27-34.
Chen JX, Li Y, Gou WL. An extracellular protease of Vibrio anguillarum: preparation of the enzyme, its physicochemical properties, gene clone and potential use in DNA vaccine. Postgraduate Conference on Marine Biology and Biotechnology, Hong Kong: 6-8 June 2001.
Chinose Y, Ehara M, Honda T. The effect on enterotoxicity of protease purified from Vibrio cholerae Ol. FEMS Microbiol Lett, 1994, 115: 265-271.
Colwell RR and Griems DJ. Vibrio diseases of marine fish populations. Helgoland. Meeresunters, 1984, 37: 265-287.
Chowdhury MAR, Miyoshi SI, Shinoda S. Vascular permeability enhacement by Vibrio mimicus protease and the mechanisms of action. Microbiol Immunol, 1991, 35: 1049-1058.
Crosa JH. A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an ironsequestering system. Nature, 1980, 284: 566-568.
Cornelis GR, Van Gijsegem F. Assembly and function of type III secretory systems. Annu Rev Micriobiol, 2000, 54: 735-74.
Dai JL, Bansal RK, Kern SE. G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4: phenotypes reversed by a tumorigenic mutation. Proc Natl Acad Sci: USA, 1999, 96 (4): 1427-1432.
Davey ML, Hancock REW, Mutharia LM. Influence of culture conditions on exprssion of the 40 kilodolton porin protein of Vibrio anguillarum serotype O2. Appl Environ Microbiol, 1998, 64: 138-246.
David VA, Deutch AH, Sloma A, Pawlyk D, Aly A, Durham DR. Cloning, sequencing and expression of the gene encoding the extracellularneutral protease, vibriolysin, of Vibrio proteolyticus. Gene, 1992, 112: 107-112.
Davis HL. Development of DNA vaccines for fish: vector design, intramuscular injection and antigene expression using viral haemorrhagic septicemia virus genes as mode. Fish Shellfish Immunol, 1998, 8: 271-286.
Denkin SM, Nelson DR. Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence. Appl Environ Microbiol, 2004, 70: 4193-4204.
Dickerson H, Clark T, Lin TL. Diagnostic and protective antigen gene sequences of Ichthyophirius multifiliis. USA: M & R 235, 00170201, 2000-02-16.
Egidius E. Vibriosis: pathogenicity and pathology, a review. Aquaculture, 1987, 67: 15-18.
Eldar A, Bejerano Y and Bercover H. Streptococcus shiloi and Streptococcus difficile: two new Streptococcal Species causing a meningoecephalitis in fish. Curr Microbiol, 1994, 28: 139-143.
Farrell DH, Crosa JH. Purification and characterization of a secreted protease from the pathogenic marine bacteria Vibrio anguillarum. Biochem, 1991, 30: 3422-3436.
Fletcher MC, Kuderova A, Cygler M. Creation of a ribonuclease abzyme through site-directed mutagenesis. Nat Bio technol, 1998, 16(11): 1065-1067.
Franco AA, Buckwold SL, Shin JW, Ascon M and Sears CL. Mutation of the Zinc-Binding Metalloprotease Motif Affects Bacteroides fragilis Toxin Activity but Does Not Affect Propeptide Processing. Infect Immun, 2005, 73: 5273-5277.
Fukasawa S, Nakamura K, Kamii A. Purification and properties of a protease from a marine luminous bacterium, Vibrio harveyi strain FLA-ll. Agri Biol Chem, 1988, 52: 435-441.
Hase CC, Finkelstein RA. Cloning and nucleotide sequence of the Vibriocholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain. J Bacteriol, 1991, 173: 3311-3317.
Helen LB, Kate FG, Steven MJ. Antibody response to yersinia pestis flantigen expressed in Salmonella typhimurium aroA from in vivo-inducible promoters. Vaccine, 2000, 18: 2668-2676.
Hensen E, Fernandez K, Goldspink G, Butterworth P, Umeda PK, Chang KC. Strong expression of foreign genes following direct injection into fish muscle. FEBS Lett, 1991, 1(290): 73-76.
Heppell J, Lorenzen N, Armstrong NK, Wu T, Lorenzen E, Einer-jensen K, Schorr J, Davis HL. Development of DNA vaccines for fish: vector design, intramuscular injectin and antigenexpression using viral haemorrhagic septicemia virus genes as model. Fish Shellfish Immonol, 1998, 8: 271-286.
Hirono I, Masuda T and Aoki T. Cloning and detection of the hemolysin gene of Vibrio anguillarum. Microbiol Pathoqenesis, 1996, 21: 173-182.
Horne MT and Baxende A. The adhesion of Vibrio anguillarum to host tissues and its roles in pathogenesis. J Fish Diseae, 1983, 6: 461-471.
Ichinose Y, Yamamoto K, Nakasome N. Enterotoxicity of El Tor-like hemolysin of non-Ol Vibrio cholerae. Infect Immun, 1987, 55: 1090-1093.
Ichinose Y, Ehara M, Honda T. The effect on enterotoxicity of protease purified from Vibrio cholerae Ol. FEMS Microbiol Lett, 1994, 115: 265-271.
Inamura H, Nakai T, Muroga K. An extracellular protease produced by Vibrio anguillarum. Bull Jpn Soc Sci Fish, 1985, 51: 1915-1920.
Ingunn S, Lorenzen E, Lorenzen N. A DNA vaccine directed against a rainbow trout rhabdovirus induces early protection against a nodavirus in turbot. Vaccine, 2003, 21: 4661-4667.
Joel H, Heather LD. Application of DNA vaccine technology to aquaculture. Adv Drug Deliver Rev, 2000, 43: 29-43.
Jorgensen JB, Johansen A, Stenersen B. CpG oligodeoxynucleotides and plasmid DNA simulate Atlantic salmon (salmo salar L.) leucocytes toproduce supernatants with antiviral activity. Dev Comp Immunol, 2001a, 25: 313-21.
Jorgensen JB, Zhou J, Johansen A. Immunostimulatory CpG oligodeoxynucleotides simulate expression of IL-1βand interferon-like cytokines in rainbow trout macrophages via a chloroquine-sensitive mechanism. Fish Shellfish Immunol, 2001b, 11: 673-82.
Kammann M, Laufs J, Schell J. Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acid Res, 1989, 17(13): 5404.
Kanemori Y, Nakai T. and Muroga K. The role of Extracellular protease produced by Vibrio anguillarum. Fish Pathology, 1987, 22(3): 153-158.
Kanno T, Nakai T and Muroga K. Scanning electron microscopy on the skin surface of ayu plecoglossus altivelis infected with Vibrio anguillarum. Dis Aquat Organ, 1990, 8: 73-75.
Karan MG, Horstedt P, Milton DL. Identification and characterisation of additional flagellin genes from Vibrio anguillarum. J Bacteriol, 1996, 178(9): 5188-5198.
Kawamoto S, Shibano Y, Fukushima J, Ishii N, Morihara K and Okuda K. Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastase against Pseudomonas infection. Infect Immun, 1993, 61: 1400-1405.
Kim SK, Yang JY, Cha J. Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04. Gene, 2002, 283: 277-286.
Kono M, Miyaza KIG, Nakamu RAH. Site-directed mutagenesis in hemoglobin: at tempts to control the oxygen affinity with cooperativity preserved.Protein Eng, 1998, 11(3): 199-204.
Kooi C, Corbett CR and Sokol PA. Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease. J Bacteriol, 2005, 187: 4421-4429.
Kothary MH, Kreger AS. Purification and characterization of an elastolytic protease of Vibrio vulnificus. J Gen Microbiol, 1987, 133: 1783-1791.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 277: 680-685.
Laird AD, Morrison DK, Shalloway D. Characterization of Raf-1 activation in mitosis. J Biol Chem, 1999, 274 (7): 4430-4439.
Laygren B, Fausa PE, Wergeland H. An extracellular low molecular weight protease with activity against gelatin and casein produced earlier during growth of Aeromonas salmonicida ssp,Salmonicida than the serine protease. Bull Euro Assoc Fish pathol, 1998, 18: 96-101.
Lee CY, Su SC, Liaw RB. Molecular analysis of an extracellular protease gene from Vibrio parahaemolyticus.Microbiol, 1995, 141: 2569-2576.
Lee JH, Kim GT, Lee TY. Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus. BBA, 1998, 1384: 1-6.
Lee CY, Cheng MF, Yu MS. Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus. FEMS Microbiol Lett, 2002, 209(1): 30-36.
Lida T and Honda T. Hemolysins produced by Vibrios.Journal of Toxicology Toxin Rev, 1997, 16: 215-227.
Lorenzen N, Lorenzen E, Einer-Jensen K. DNA vaccines as a tool for analyzing the protective immune response against rhabdoviruses in rainbow trout. Fish Shellfish Immunol, 2002a, 12: 439-453.
Lorenzen N, Lorenzen E, Katja E J. Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens. Developmental and comparative Immunology, 2002b, 26: 173-179.
Lorenzen N, Niels JO, Claus K. Immunity to VHS virus in rainbow trout. Aquaculture, 1999, 172: 41-61.
Lorenzen N, Lorenzen E, Katja EJ. Immunity to viral haemorrhagic septicaemia (VHS) following DNA vaccination of rainbow trout at an early life-stage. Fish & Shellfish Immunology, 2001, 11: 585-591.
Kanellos TS, Sylvester ID. Mammalian granulocytemacrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish. Immunology, 1999, 96: 507-510.
Kristine R, Beate JT, Oystein E. Expression of luciferase in selected organs following delivery of naked and formulated DNA to rain bow trou t (Oncorhynchus mykiss) by different routes of administration. Fish Shellfish Immunol, 2004, 16: 251-264.
Kumar SR, Parameswaran V, Ishaq Ahmed VP, Musthaq SS, Sahul Hameed AS. Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum. Fish Shellfish Immunol, 2007 Aug, 23(2): 316-26.
Maeda H. Role of microbial protease in pathogenesis. Microbiol Immunol, 1996, 40: 685-699.
Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selectior. Proc Natl Acad Sci, USA: 1985, 82 (2): 488-492.
Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Micriobiol Rev, 1996, 60: 512-538.
Mergulhao FJM, Monteiro GA, Cabral JMS, Taipa MA. Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. J Microbiol Biotechnol, 2004, 14: 1-14.
Marta A, Marc J, Ben S. A fish specific expression vector containing the interferon regulatory factor 1A (IRF1A) promoter for genetic immunization of fish. Vaccine, 2003, 21: 1591-1600.
Maureen KP, Gael K, Kyle AG. Quantitative expression profiling of immuneresponse genes in rainbow trout following infectious haematopoietic necrosis virus (IHNV) Infection or DNA vaccination. Fish & Shellfish Immunology, 2004, 17: 447-462.
McIver K, Kessler E and Ohman DE. Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase. J Bacteriol, 1991, 173: 7781-7789.
McIver KS, Olson JC and Ohman DE. Pseudomonas aeruginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing, and secretion. J Bacteriol, 1991, 175: 4008-4015.
Michael JM, Heather LD. CpG DNA as mucosal adjuvant. Vaccine, 2000, 18: 231-237.
Mikalsen AB, Sindre H, Torgersen J, Rimstad E. Protective effects of a DNA vaccine expressing the infectious salmon anemia virus hemagglutinin-esterase in Atlantic salmon. Vaccine, 2005, 23: 4895-4905.
Milton DL, Norqvist A, Wolf-Watz H. Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum. J Bacteriol, 1992, 174: 7235-7244.
Milton DL, Norqvist A, Wolf-Watxtz H. Sequence of a novel virulence-mediating gene, virC, from Vibrio anguillarum. Gene, 1995, 164(1): 95-100.
Milton DL, O' Toole R, Horstedt P, Wolf-Watz H. Flagellin A is essential for the virulence of Vibrio anguillarum J Baoteriol, 1996, 178(5): 1310-1319.
Miyoshi S, Sugiyama K, Suzuki Y. Enhancement of vascular permeability due to histamine-releasing effect of Vibrio vulnificus protease. FEMS Microbiol Lett, 1987, 40: 95-98.
Miyoshi S, Wakae H, Tomochika. Functional domains of a zinc metalloprotease from Vibrio vulnificus. J Bacteriol, 1997, 179: 7606-7610.
Miyoahi S, Nakazawa H, Tomochika K. Characterization of the hemmorrhagic reaction caused by Vibrio vulnificus metallopretease, a member of the thermolysin family. Infect Immun, 1998, 66: 4851-4855.
Miyoshi DL, Nakazawa H, Kawata K. Characterization of the hemorrhagic reaction caused by Vibrio vulnificus metalloprotease, a member of the hemolysin family. Infect Immune, 1998, 66: 4851-4855.
Mo ZL, Tan XG, Xu YL. A Vibrio anguillarum strain associated with ulcerative skin in cultured flounder, Paralichthys olivaceus. Chin J Oceano Lumino, 2001, l19(4): 319-326.
Montero AB and Austin B. Characterization of extracellular products from an isolate of Vibrio harveyi recovered from diseased post-larval Penatus vannamei (Bonne). J Fish Dis, 1999, 22: 377-386.
Mukhopadhya YP, Roy KB. Protein engineering of BamHI restriction endonuclease: replacement of Cys54 by Ala enhances catalytic activity. Protein Eng, 1998, 11(10): 931-935.
Munn CB. Haemolysin production by Vibrio anguillarum. FEMS Microbiol Lett, 1978, 3: 265-268.
Mun CB. Production and properties of a haemolytic toxin by Vibrio anguillarum. Fish Diseases. Third copraq session, Springer-Verlag, Berlin: 1980, 69-74.
Muroga K. Viral and bacterial diseases of marine fish and shellfish in Japanese hatcheries. Aquaculture, 2001, 202: 23-24.
Nakamura Y, Nakai T, Muroga K. Induction of antibody producing cells in some fishes by immunization with Vibrio anguillarum O-antigen. Fish Pathol, 1990, 25: 225-230.
Nieto TP and Ellis AE. Characterization of extracellular metalo-and serine-protease of Aeromonas hydrophila strain B51. Journal of Gen Microbiol, 1986, 132: 1975-1979.
Nishibuchi M and Kaper JB. Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus. J Bacteriol, 1985, 162:558-564.
Nishibuchi M, Khaeomaneeiam V, Honda T. Comparative analysis of the hemolysin qenes of Vibrio cholerae non-Ol, V. mimicus,and V. hollisae that are similar to the tdh gene of V. parahaemolyticus. FEMS Microbiol Lett, 1990, 67: 251-256.
Noonan B, Enzmann PJ, Trust TJ. Recombinant infectious hematopoietic nercosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromanas salmonicida induce protective immunity in rain trout (Oncorhynchus mykiss). Appl Environ Microbiol, 1995, 61(10): 3586-3591.
Norqvist A, Norrman B, Wolf-Watz H. Identification and characterization of a zinc metalloprotease associated with invasion by the fish pathogen Vibrio anguillarum. Infect Immun, 1990, 58: 3731-3736.
Norqvist A, Wolf-Watxtz H. Characterization of a novel chromosomal virulence locus involved in expression of a major surface flagellar sheath antigen of the fish pathogen Vibrio anguillarum. Infect Immun, 1993, 61(6): 2434-2444.
Olsson JC, Joborn A, Westerdahl A. Is the turbot, Scophthalmus maximus(L), intestine a partial of entry for the fish pathogen Vibrio anguillarum. J Fish Disease, 1996, 19: 225-234.
Ormonde P, Hoerstedt P, O’Toole R. Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum. J Bacteriol, 2000, 182(8): 2326-2328.
Pathiratne A, Widauapathiraua G.S and Chandrosrkanthi WHS. Association of Aeromonas hydrophila with epizootic ulcerative syndrome (EUS) in freshwater fish in Sri Lanka. Journal Appl Ichthyol, 1994, 10: 204-208.
Pavlovskis OR, Wretlind B. Assessment of protease (elastase)as a Pseudomonas aeruginosa virulence factor in experimental mouse burn infection. Infect Immun, 1979, 24: 181-187.
Pederson K, Gram L, Austin DA. Pathogenicity of Vibrio anguillarum serogroup Ol compared to plasmids,outer membrane protein profile,and siderophore production. J Appl Microbiol, 1997, 82: 365-371.
Pyle SW and Shotts EB. A new approach for differiating flexihacferia isolated from cold-water and warm-water fish. Can J Fish Aquat Sci, 1980, 37: 1040-1042.
Ransom DP. Bacteriologic immunologic and pathologic studies of Vibrio sp. pathogenic to salmonids. Ph.D.thesis, Oregen State University, Corvallis: 1978.
Rawlings ND and Barrett AJ. Evolutionary families of metallopeptidases. Methods Enzymol, 1995, 248: 183-228.
Salati F, Watanabe K, Kawai K et al. Immune response of ayu against Vibrio anguillarum Lipopolysaccharide, Nippon Suisan Gakkaishi,1989,55:45-49
Seo JY, Kim KH, Kim SG, Oh MJ, Nam SW, Kim YT, Cho TJ. Protection of flounder against hirame rhabdovirus (HIRRV) with a DNA vaccine containing the glycoprotein gene. Vaccine, 2006, 24: 1009-1015.
Shinoda S. Protein toxins produced by pathogenic vibrios. Journal of Natural Toxicon, 1999, 8: 259-269.
Simon M, Mathes A, Blanch A. Characterization of a porin from the outer membrane of Vibrio anguillarum. J Bacteriol, 1996, 178: 4182-4188.
Singer J T, Schmidt K A, and Reno P W. Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virolence of the marine fish pathogen Vibrio anguillarum. Bacteriology, 1991, 173: 1347-1352.
Suzuki S, Kuroe K, Yasue K. Antigenicity and N-terminal amino acid sequence of a 35 kDa porin-like protein of Listonella (vibrio) anguillarum: comparison among different serotypes and other bacterial species. Lett Appl Microbiol, 1996, 23(5): 303-306.
Takeuchi H, Shibano Y, Morihara. Structural gene and complete amino acidsequence of Vibrio alginolyticus collagenase. Biochem J, 1992, 281: 703-708.
Terai A, Shirai H, Yoshida O. Nucleotide sequence of the thermostable direct hemolysin qene(tdh gene)of Vibrio mimicus and its evolutionary relationship with the tdh genes of Vibrio Parahaemolyticus. FEMS Microbiol Lett, 1990, 71: 319-324.
Thune RL, Graham TE, Riddle LM. Extracellular proteases from Aeromonas hydrophila. partial purification and effects on aqeochannel catfish. Transact Am Fish soc, 1982, 111: 749-754.
Tiziano V, Laura I, Rita C. Assessment of DNA vaccine potential for gilthead sea bream (Sparus aurata) by intramuscular injection of a reporter gene. Fish Shellfish Immunol, 2003, 15: 283-295.
Tolmaskg ME and Crosa JH. Iron transport genes of the pJMl-mediated iron uptake system of Vibrio anguillarum are included in a transposon like strcture. Plasmid, 1995, 37: 180-190.
Tomokazu T, Akiko I, Ikuo H. Development of a DNA vaccine against hiramerhabdovirus and analysis of the expression of immune-related genes after vaccination. Fish & Shellfish Immunology, 2004, 17: 367-374.
Tsunasawa S, Suginara A, Masaki T. Amino acid sequence of termostable direct hemolysin produced by Vibrio Parahaemolyticus. J Bioch, 1987, 101: 111-121.
Tuomanen El. Cell adhesion molecules in the development of bacterial infections in leukocyte adhesion basic and clinical aspects. (Gamberg CG, Mandrap-PoulsenT and Wogensenbach L eds), Elsevier Sciencc Publishers, New York: 1992, 297-306.
Ulmer JB, Donnely J J, Parker SE. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 1993, 59: 1745-1749.
von Heijne G. A new method for predicting signal sequence cleavage sites.Nucleic Acids Res, 1986, 14: 4683-4690.
Wane A, Pakl KA. Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability. Biochem, 1998, 37(38) : 13120- 13127.
Wang XH, Oon HGW. Internalization and cytotoxicity are important virulence mechanisms in vibrio-fish epithelial cell interactions. Micro, 1998, 144(11): 2987-3002.
Wolff IA, Malone RW, Williams P. Genetic immunization is a simple method for eliciting an immune respones. Nature, 1992, 356: 152-154.
Wolke RE. Pathology of bacteriol and fungal disease affecting fish In:Ribelin W E and Migaki G(eds), The Pathology of Fishes. Wisconsin: University of Wisconsin Press, 1975: 76-78.
Yamamoto K, Ichinose Y, Shinagawa H. Two-step processing for activation of the cytolysin/ hemolysin of the structural gene (hlyA) and characterization of the processed products. Infect Immun, 1990, 58: 4106-4116.
Yamasoki S, Shirai H, Takeda Y. Analysis of the qene of Vibrio hollisae encoding the hemolysin similar to the thermostable direct hemolysin of Vibrio parahaemolyticus. FEMS Microbiol Lett, 1991, 80: 259-264.
Yi AK, Tuetken R, Redford T. CpG motifs in bacterial DNA activate oxygenspecied. Immunol, 1998, 160: 4755-4761.
Zhen M, Jianzhong S, Lixin X. CpG oligodeoxynucleotides activate grass carp (ctenopharyngo don idellus) macrophages. Dev Com Immunol, 2003, 27: 313-321.
Zheng FR, Sun XQ, Liu HX, Zhang JX. Study on the distribution and expression of a DNA vaccine against lymphocystis disease virus in Japanese flounder(Paralichthys olivaceus). Aquaculture, 2006, 261: 1128-1134.