摘要
海水养殖鱼类细菌性疾病是一类常见且危害大的疾病,限制了海水养殖业的可持续发展。为解决养殖鱼类的病害问题,增强养殖品种的抗病力,必须对病原菌的致病因子、鱼类的免疫能力以及病原菌和鱼类免疫器官之间的相互作用关系进行全面地了解。大菱鲆(Scophthalmus maximus)的弧菌病是一种危害性较大的细菌性疾病,国内外对大菱鲆的弧菌病进行了较多的研究,但对病原菌和大菱鲆免疫器官相互作用关系的实时研究较少,许多免疫相关基因尚不清楚。研制绿色生物渔药和利用免疫相关基因进行抗病育种是促进我国水产养殖业可持续发展的有效尝试途径,因此必须对病原菌致病机理及鱼类免疫能力进行全面深入的研究。
本研究利用已构建的感染哈维氏弧菌(Vibrio harveyi)的大菱鲆的头肾和脾脏差减cDNA文库,探索大菱鲆与哈维氏弧菌相互作用的实时特征。采用腹腔注射法分别向35尾大菱鲆注射0.15 ml (3×107CFUs/ml)的哈维氏弧菌菌液,同时设置注射等量生理盐水和未注射的对照组(每组各4尾)；分别取哈维氏弧菌感染后4、8、12、24、36 h的活鱼(每组4尾),注射生理盐水8h后的对照组以及未注射组的大菱鲆进行解剖,取头肾、肾、心脏、肝脏、肠、肌肉、脾和鳃组织并提取总RNA以备后期RACE扩增及表达分析使用；利用RACE技术获得了4种免疫相关基因的全长,并使用生物信息学方法进行了理化性质分析、结构分析和同源性分析；用实时荧光定量PCR技术研究了免疫相关基因在不同组织中的表达以及受哈维氏弧菌感染前后的表达变化。另外,还探讨了溶藻弧菌(Vibrio alginolyticus)的毒力因子TLH溶血素对鱼类的致病性。
1.大菱鲆cathepsin D基因的克隆及表达分析
Cathepsin D是溶酶体内的一种天冬氨酸蛋白水解酶,同时也存在于巨噬细胞的内涵体中,在生物体的发育及生理过程中起着重要的作用。本研究获得了大菱鲆cathepsin D的全长cDNA,其ORF为1191 bp,在预测的氨基酸序列中存在由18个氨基酸组成的信号肽和由43个氨基酸组成的前导肽。经氨基酸同源比对发现,大菱鲆cathepsin D与其他物种有高度的同源性,与尖吻鲈(Lates calcarifer)的相似性达89%。荧光定量表达分析显示cathepsin D在肝脏中表达量最高。受哈维氏弧菌感染后,cathepsin D在肝脏、脾脏和头肾中的表达量均在8h达到最低。此后,在肝脏和头肾中cathepsin D的表达量又逐步上升,并在24 h超过了初始表达量；cathepsin D在脾脏中的表达量从12h起就高于初始表达量并维持这一表达水平至36h。根据研究结果推测cathepsin D可能具有抗原呈递的作用,而且在感染12h后可能参与了细胞的凋亡过程。
2.大菱鲆CXCR4基因的克隆及表达分析
CXCR4属于G蛋白偶联受体超家族的一员。本研究获得的大菱鲆CXCR4 cDNA全长为1349 bp,编码372 aa。一级结构分析显示大菱鲆CXCR4与其他脊椎动物的序列具有高度相似性。大菱鲆CXCR4在脾脏组织中的表达量最多。感染后,CXCR4在脾脏和头肾的表达量分别于8h和12h出现最低值,之后又都逐渐上升直至36h。
3.大菱鲆Rac1基因的克隆及表达分析
Rac1是Rho小GTP结合蛋白家族中的成员。它是调节细胞骨架肌动蛋白的重要信号分子,可以间接地控制细胞的形态及运动。本研究获得了2420bp的大菱鲆Rac1全长cDNA序列,预测的氨基酸序列与其他Rac亚家族中的成员有高度相似性,与人类Rac1的相似性达95.3%。荧光定量分析显示,大菱鲆Rac1在所有组织中都有组成型表达。免疫感染后,Rac1在肝脏中的表达较为波动,分别在8h和24 h出现了两次上调表达,在12h和36 h出现两次下调表达。在头肾中,Rac1在4h的表达出现下降,之后在24 h回升至初始表达水平。
4.大菱鲆hsc70基因的克隆及表达分析
大菱鲆HSC70的全长cDNA序列包含一个1956 bp的开放阅读框。在预测的651个氨基酸序列中发现了Dnak特征性基序,胞质HSP70特征基序以及四肽简并重复序列,而且与其他真核生物HSP70家族成员具有较高同源性。表达分析显示,hsc70在大菱鲆各组织中呈组成型表达；受哈维氏弧菌感染后,大菱鲆肝脏和脾脏中的hsc70表达水平分别在24 h和12h出现上调(2.5倍和1.6倍)；注射生理盐水组与未注射组之间差异不显著。
5.溶藻弧菌溶血素tlh的克隆表达、蛋白纯化及致病性研究
溶血素是许多病原菌的致病因子。本研究克隆表达了溶藻弧菌溶血素的tlh基因(ORF为1254 bp),其预测氨基酸序列含有GDSL磷脂酶结构域。将该基因在大肠杆菌中重组表达后,利用Ni琼脂糖亲和柱进行纯化,纯化蛋白具有磷脂酶活性和溶血活性,其溶血活性为18 HU/μg。活性分析显示,二价金属离子对TLH的溶血活性都有不同程度的抑制,而一价金属离子则不影响其活性。TLH的溶血活性受到蛋白质化学修饰剂的强烈影响。致病性研究显示,TLH对牙鲆鳃细胞和斑马鱼具有较强的毒性及致病性,对斑马鱼的半数致死剂量为0.8μg/g鱼。对溶藻弧菌致病因子TLH的研究,可以促进利用毒力因子作为抗原的疫苗开发,还可以为弧菌病提供检测方法。
本论文在分子水平上阐述了鱼类免疫系统和病原菌之间的相互作用,阐明了溶藻弧菌的溶血素在其致病性中的作用。该研究为鱼类品种改良和病害防治奠定了基础,也为研究弧菌及其毒力因子与海鱼之间的相互作用关系提供了基础知识。
Bacterial diseases are serious problems in marine cultured fish, which is a limiting factor to aquaculture. In particular, Vibrio harveyi is a bacterial pathogen responsible for serious disease outbreaks in numerous species of cultured fish including turbot (Scophthalmus maximus). Many researches have been done in vibriosis of turbot in China and abroad. However, only a few immune-related genes, which could be used in fish breeding for disease resistance, have been isolated and characterized in turbot. Therefore, it is necessary to elucidate the virulence factors of pathogens, the immunity of fish and the interaction between Vibrio and the immune organs of fish.
In previous study, suppression subtractive hybridization (SSH) was used to investigate the response of turbot to V. harveyi, and a cDNA library from kidney and spleen of artificially infected turbot was constructed. In this study, the bacterial suspension of 3×107 CFUs/ml was injected intraperitoneally in 0.15 ml volumes into a group of 35 turbot. In parallel, a group of 4 fish were injected with physiological saline (PS) as controls, and another group of 4 fish were non-injected as blank controls. Sub-groups of 4 bacterial-infected fish were sacrificed at 4,8,12,24 and 36 h. The control fish were killed after 8 h. Samples of head kidney, kidney, heart, liver, intestine, muscle, spleen and gill were collected, and total RNA were extracted with Trizol reagent. Four immune-related genes were cloned and characterized, and their expressions were analyzed in different tissues of turbot using quantitative real-time PCR (qPCR) at different times after challenged with V. harveyi. In addition, the TLH hemolysin from Vibrio alginolyticus was expressed, purified and characterized.
Cathepsin D is a lysosomal endoproteolytic aspartic proteinase which also has been found in endosomes of macrophage. It is thought to play key roles in the developmental and physiological process of animals. In the present study,5'-RACE and 3'-RACE were carried out to obtain the complete cDNA sequence of turbot cathepsin D, which contained a 1191 bp open reading frame (ORF). The deduced amino acid sequence of the cathepsin D consisted of a signal peptide of 18 aa, a leader peptide extending 43 aa, and a mature peptide of 335 aa. BLAST analysis revealed that turbot cathepsin D shared high similarity with other known cathepsin D, and it showed significant homology with that of Barramun (Lates calcarifer B., 89% aa similarity). qPCR demonstrated that the highest expression level of the turbot cathepsin D was in liver. After turbot were challenged with V. harveyi, the lowest expression levels of cathepsin D in liver, spleen and head kidney were detected at 8 h. The expression levels of cathepsin D in liver and head kidney increased gradually after 8 h and exceeded the background level after 24 h. In spleen, the expression level was reinforced after 8 h and kept at the level that was higher than the original level after 12 h. The results suggested that cathepsin D might process antigens for presentation to the immune system and have synergetic effect with apoptosis pathway until 12 h after injection.
Chemokine receptor 4 (CXCR4) belongs to the large superfamily of G protein-coupled receptors. The full-length cDNA sequence of turbot CXCR4 was obtained, and sequence analysis indicated that its primary structure was highly similar to CXCR4 from other vertebrates. qPCR demonstrated that the highest expression level of turbot CXCR4 was in the spleen following injection with PS. After turbot were challenged with V. harveyi, the lowest expression level of CXCR4 was detected at 8 h in the spleen and 12 h in the head kidney, and then increased gradually to 36 h.
Racl is a small GTP-binding protein belongs to the Rho small GTPases, which are important signaling molecules that regulate the dynamics of the actin cytoskeleton and mediate changes in cell morphology and motility. The full-length cDNA sequence of turbot Racl has 2420 nucleotides (nt) encoding a protein of 192 amino-acids. At the amino-acid level, turbot Racl was highly conserved to previously characterized GTPases of Rac sub-family, and was nearly identical to human Rac1 (95.3% identity). qPCR demonstrated that the Racl was constitutively expressed in all tissues examined, but at different levels. Upon challenge with V. harveyi, the expression level of Racl fluctuated in the liver at different time points. In the head kidney, its expression level decreased to the lowest at 4 h, and then increased to the background level at 24 h.
HSC70 is a constitutively expressed member of the 70 kDa class of HSP70, which plays key roles in the cell as molecular chaperone and involves in a number of cellular processes. The full length HSC70 cDNA of 2188 bp contained a 1956 bp ORF encoding 651 amino acids. The specific motif of Dnak (DLGTT-S-V,10—18 aa), EEVD (648—651 aa) and GGMP repeated tetra-peptide (615—630 aa) were found in the deduced amino acid sequence of turbot HSC70. Turbot HSC70 had high identity with HSC70 from other organisms. qPCR demonstrated that hsc70 mRNA expressed constitutively in all of the test tissues. After turbot were challenged with V. harveyi, the expression levels of hsc 70 mRNA were up-regulated in liver (2.5-fold) and spleen (1.6-fold) at 24h and 12h, respectively (p<0.05).
Hemolysin is a putative pathogenicity factor in many bacterial pathogens. In this study, a DNA fragment containing the open reading frame (1254 bp) of thermolabile hemolysin gene (tlh) from V. alginolyticus V05 was amplified, and cloned into an expression plasmid pET-24d (+). The deduced amino acids contained a GDSL lipase domain like VHH. The purified TLH, by Ni-NTA His-Bind Resin, showed phospholipase activity on egg yolk emulsion plate and hemolytic activity against flounder erythrocyte with the specific activity of 18 HU/μg. The addition of divalent cations at different concentrations decreased hemolytic activity of the purified TLH, but monavalent cations did not affect hemolytic activity. The hemolytic activity of TLH was also inhibited markedly by protein modification reagents, i.e.βME, PMSF and DTNB. Moreover, TLH was toxic to flounder gill (FG) cell lines, and pathogenic to zebrafish when injected intraperitoneally, with an LD50 dose of 0.8μg/g fish. This work provides a probability for developing vaccine with virulence factor and a diagnostic tool for vibriosis.
This thesis has elucidated the relationship between the immune system of fish and the pathogens, and the toxicity of TLH hemolysin of V. alginolyticus, which is a foundation for breeding of fish and disease control.
引文
陈怀清.从比较免疫学看鱼类的免疫特性.动物学杂志,1994,29(4)：56-60.
陈吉祥,李彩风,颜显辉,等.大菱鲆病原鳗弧菌生物学及分子特征研究.高技术通讯,2005,15(6)：372-375.
陈立祥,张学文,肖调义,等.人α-干扰素基因在转基因草鱼中的表达.湖南农业大学学报(自然科学版),2001,27(3)：177-178.
程顺峰.溶藻弧菌疫苗对牙鲆免疫效果的研究.青岛农业大学学报(自然科学版),2009,26(2)：124-127.
范文辉,黄健,王秀华,等.养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析.微生物学报,2005,5.
黄万红.九孔鲍养殖中脓疱病病因分析和药物敏感性研究.台湾海峡,2005,24(45)：526-532.
胡亮,赵凤梅,于兰萍,等.副溶血弧菌对养殖大菱鲆致病性研究.水产科学,2008,27(7)：340-343.
焦传珍,王在照,李富花,等.编码中国明对虾(Fenneropenaeus chinensis)一种组成型热休克蛋白70(HSC70)的cDNA克隆、测序及其表达分析.科学通报,2004,49(21)：2178-2186.
金珊,王国良.赵青松,等.海水网箱养殖大黄鱼弧菌病的流行病学研究.水产科学,2005,24(1)：17-19.
刘峰,李家乐.草鱼免疫因子研究进展.上海海洋大学学报,2009,18(4)：502-507.
刘广锋,周世宁,徐力文,等.杂色鲍幼苗“急性死亡脱落症”病原菌分析.中国水产科学,2006,13(4)：655-661.
刘洋.大菱鲆(Scophthalmus maximus)5种趋化因子的克隆、鉴定及表达分析：[博士学位论文].青岛：中国海洋大学,2007.
刘毅,隗黎丽,李莉,等.福尔马林灭活柱状黄杆菌对草鱼免疫相关基因表达的影响.水生生物学报,2008,32(6)：794-801.
龙华,郑英.鱼类抗病基因的研究进展.中国医学研究与临床,2006,4(1)：32-47.
卢全章.草鱼胸腺组织学的研究.水生生物学报,1991,15(4)：327-331.
马爱军,雷霁霖,陈四清,等.大菱鲆营养需求与饲料研究进展.海洋与湖沼,2003,34(7)：450-459.
倪纯治,林燕顺,叶德赞.大亚湾和厦门湾的嗜盐弧菌.海洋学报,1995,17(5)：124-129.
牛化欣,苏跃朋,苏志良.我国北方地区几种新开发的海水养殖经济鱼类及其发展状况.中 国水产,2006,11：30-33.
潘学霞,邵健忠,项黎新,等.鱼类中几种新型免疫因子的进展.水产学报,2005,29(2)：264-269.
彭悟,汤雅男,胡成钰CaPKR-like在鲫鱼与草鱼组织中的表达特性分析.动物学研究,2007,28(5)：465-469.
彭喜春,张宁,冉艳红,等.海产品中溶藻弧菌的筛选及其致病因子的研究.现代食品科技,2008,24(4)：312-315.
史成银,王印庚,秦蕾,等.我国养殖大菱鲆病毒性红体病及其流行情况调查.海洋水产研究,2005,26(1)：1-6.
万文菊,王纪亭,石存斌,等.溶藻弧菌感染对剑尾鱼HSP70基因表达的影响.大连水产学院学报,2007,22(5)：330-334.
王蓓,黄郁葱,汤菊芬,等.溶藻弧菌生物被膜的特性与相关基因的研究.安徽农学通报,2009,15(8)：29-31.
王海芳,孙际佳,赵典惠,等.溶藻弧菌疫苗对黑鲷的免疫效果研究.华南农业大学学报,2008,29(1)：97-100.
王小红,谢笔钧,史贤明,等.金黄色葡萄球菌B型肠毒素的培养产毒与柱层析纯化.食品科学,2005,26(5)：88-91.
王印庚,秦蕾,张正,等.养殖大菱鲆的爱德华氏菌病.水产学报,2007,31(4)：487-495.
王印庚,陈洁君,秦蕾.养殖大菱鲆蟹栖异阿脑虫感染及其危害.中国水产科学,2005,12(5)：594--601.
吴海峰,白俊杰,劳海华,等.草鱼Mx蛋白体内诱导表达的初步研究Anim Biotechnol Bull, 2004,19(1):12-17.
吴后波,胡超群.合浦珠母贝体内细菌种类组成的初步研究.南海研究与开发,1996,2：58-60.
谢海侠,聂品.鱼类胸腺研究进展.水产学报,2003,27(1)：90-96.
徐怀恕,杨学宋,李筠,等.对虾苗期细菌病害的诊断与控制.北京：海洋出版社,1999.
闫茂仓,陈少波,单乐州,等.海水养殖动物致病弧菌的研究进展.水产科学,2009,28(8)：475-481.
杨先乐.鱼类免疫学研究进展.水产学报,1989,13(3)：272-284.
于燕.半滑舌鳎(Cynoglossus semilaevis) TLR9及其信号传导相关基因的克隆与表达：[博士学位论文].青岛：中国海洋大学,2009.
张生元,艾桃山,喻运珍,等.草鱼3种不同类型肝损伤模型的初步建立.内陆水产,2009, 9：42-45.
张晓华.海洋微生物学.青岛：中国海洋大学出版社,2007.
张晓君,房海,陈翠珍,等.大菱鲆爱德华氏菌病：病例报告.中国兽医学报,2007,4.
张义兵,张奇亚,桂建芳.鱼类的干扰素系统和干扰素系统基因的鉴定.水生生物学报,2004,28(3)：317-322.
张永安.鱼类免疫组织和细胞的研究概况.水生生物学报,2000,24(6)：648-652.
张占会,张其中,李春勇,等.养殖近江牡蛎致病弧菌的分离与鉴定.热带海洋学报,2008,27(6)：49-56
郑国兴,李何,黄宁宇,等.文蛤病原茵(溶藻弧菌)的分离与性状及病文蛤组织的电镜观察.水产学报,1991,15(2)：85-95.
钟明超,黄浙.鲇鱼淋巴样器官的发育.水产学报,1995,19(3)：262-327.
周丽,战文斌,俞开康.海水养殖鱼类病害发展现状及控制措施.齐鲁渔业,2002,19(5)：28-30.
邹玉霞,张培军,莫照兰,等.大菱鲆出血症病原菌的分离和鉴定.高技术通讯,2004,14(4)：89-93.
Ackerman PA, Iwama GK. Physiological and cellular stress responses of juvenile rainbow trout to vibriosis. J Aquat Anim Health,2001,13(2):173-180.
Agarraberes FA, Terlecky SR, Dice JF. An intralysosomal HSP70 is required for a selective pathway of lysosomal protein degradation. J Cell Biol,1997,137(4):825-834.
Alabyev BY, Najakshin AM, Mechetina LV, et al. Cloning of a CXCR4 homolog in chondrostean fish and characterization of the CXCR4-specific structural features. Dev Comp Immunol, 2000,24:765-770.
Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSI-BLAST:a new generation of protein database search programs. Nucleic Acids Res,1997,25:3389-3402.
Amer AO, Swanson MS. Autophagy is an immediate macrophage response to Legionella pneumophila. Cell Microbiol.2005,7:765-778.
Aoki T, Hirono I. Immune relevant genes of Japanese flounder, Paralichthys olivaceus. Comp Biochem Phys D,2006,1:115-121.
Austin B, Austin DA. Methods for the microbiological examination of fish and shellfish. UK:Ellis Horwood, Chichester,1989:99-140.
Austin B, Pride C, Rhodie GA. Association of a bacteriophage with virulence in Vibrio harveyi. J Fish Dis,2003,26:55-58.
Bakkers J, Kramer C, Pothof J, et al. Has2 is required upstream of Racl to govern dorsal migration of lateral cells during zebrafish gastrulation. Development,2004,131:525-537.
Baoprasertkul P, He CB, Peatman E, et al. Constitutive expression of three novel catfish CXC chemokines:homeostatic chemokines in teleost fish. Mol Immunol,2005,42:1355-1366.
Basmaciogullari S, Pacheco B, Bour S, et al. Specific interaction of CXCR4 with CD4 and CD8: functional analysis of the CD4/CXCR4 interaction in the context of HIV-1 envelope glycoprotein mediated membrane fusion. Virology,2006,353:52-67.
Bayne CJ, Gerwick L, Fujiki K, et al. Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means Suppression Subtractive Hybridization. Dev Com Immunol,2001,25(3):205-217.
Berchem GJ, Glondu M, Gleizes M, et al. Cathepsin-D affects multiple steps of tumor progression: Proliferation, angiogenesis and apoptosis. Oncogene,2002,51:5951-5955.
Bird S, Zou J, Secombes CJ, et al. Evolution of interleukin-1 beta gene in vertebrates. Cytokine Growth Factor Rev.2002,13(6):483-502.
Bokoch GM. Regulation of innate immunity by Rho GTPases. Trends. Cell Biol,2005,15: 163-171.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem,1976,72:248-254.
Brooks S, Tyler CR, Carnevali O, Coward K, Sumpter JP. Molecular characterization of ovarian cathepsin D in the rainbow trout, Oncorhynchus mykiss. Gene,1997,201:45-54.
Busillo JM, Benovic JL. Regulation of CXCR4 signaling. Biochim Biophys Acta,2007,1768: 952-963.
Cai SH, Wu ZH, Jian JC, et al. Cloning and expression of gene encoding the thermostable direct hemolysin from Vibrio alginolyticus strain HY9901, the causative agent of vibriosis of crimson snapper (Lutjanus erythopterus). J Appl Microbiol,2007,103:289-296.
Capasso C, Lees WE, Capasso A, et al. Cathepsin D from the liver of the Antarctic icefish Chionodraco hamatus exhibits unusual activity and stability at high temperatures. BBA-Protein Struct M,1999,1431:64-73.
Castex M, Lemaire P, Wabete N, et al. Effect of probiotic Pediococcus acidilactici on antioxidant defences and oxidative stress of Litopenaeus stylirostris under Vibrio nigripulchritudo challenge. Fish Shellfish Immun,2010,28(4):622-31.
Carnevali O, Centonze F, Brooks S, et al. Molecular cloning and expression of ovarian cathepsin D in seabream, Sparus aurata. Biol Reprod,1999,61:785-791.
Chaves-Pozo E, Munoz P, Lopez-Munoz A, et al. Early innate immune response and redistribution of inflammatory cells in the bony fish gilthead seab-ream experimentally infected with Vibrio anguillarum. Cell Tissue Res,2005,320:61-68.
Cheret R, Delbarre-Ladrat C, Lamballerie-Anton M, et al. Calpain and cathepsin activities in post mortem fish and meat muscles. Food Chem,2007,101:1474-1479.
Chuang K, Ho S, Song Y. Cloning and expression analysis of heat shock cognate 70 gene promoter in tiger shrimp (Penaeus monodon). Gene,2007,405:10-18.
Cripe TP, Delos SE, Estes PA, et al. In vivo and in vitro association of hsc70 with polyomavirus capsid proteins. J Virol,1995,69:7807-7813.
Cuervo AM, Dice JF. Lysosomes, a meeting point of proteins, chaperones, and proteases. J Mol Med,1998,76:6-12.
Currie S, Tufts B. Synthesis of stress protein 70 (HSP70) in rainbow trout (Oncorhynchus mykiss) red blood cells. J Exp Biol,1997,200(3):607-614.
Cyster JG. Chemokines and cell migration in secondary lymphoid organs. Science,1999,268: 2098-2101.
Dambly-Chaudiere C, Cubedo N, Ghysen A. Control of cell migration in the development of the posterior lateral line:antagonistic interactions between the chemokine receptors CXCR4 and CXCR7/RDC1. BMC Dev Biol,2007,7(23).
Daniels GD, Zou J, Charlemagne J, et al. Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbow trout Oncorhynchus mykiss. J Leukoc Biol,1999,65:684-690.
Deane EE, Woo NYS. Cloning and characterization of the hsp70 multigene family from silver sea bream:Modulated gene expression between warm and cold temperature acclimation. Biochem Bioph Res Co,2005,330:776-783.
de Duve C. Lysosomes revisited. Eur J Biochem,1983,137:391-397.
Dell'Angelica EC, Mullins C, Caplan S, et al. Lysosome-related organelles. FASEB J,2000,14: 1265-1278.
Demand J, Luders J, Hohfeld J. The carboxy-terminal domain of HSC70 provides binding sites for a distinct set of chaperone cofactors. Mol Cell Biol,1998,18(4):2023-2028.
Diatchenko L, Lau YF, Campbell AP, et al. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specitic cDNA probes and libraries. Proc Natl Acad Sd USA,1996,93(12):6025-6030.
Diment S, Martin KJ, Stahl PD. Cleavage of parathyroid hormone in macrophage endosomes illustrates a novel pathway for intracellular processing of proteins. J Biol Chem,1989,264: 13403-13406.
Dreyer RN, Bausch KM, Fracasso P, et al. Processing of the preamyloid protein by cathepsin D is enhanced by a familial Alzheimer's disease mutation. Eur J Biochem,1994,224:265-271.
Dutra WO, Moreira PR, Souza PEA. Implications of cytokine gene polymorphisms on the orchestration of the immune response:Lessons learned from oral diseases. Cytokine Growth F R,2009,20:223-232.
Egidius E. Vibriosis:pathogenicity and pathology. A review. Aquaculture.1987.67:15-28.
Ellis AE. Innate host defence mechanism of fish against viruses and bacteria. Dev Comp Immunol, 2001,25:827-839.
Faber A, Roderburg C, Wein F, et al. The many facets of SDF-1α, CXCR4 agonists and antagonists on hematopoietic progenitor cells. J Biomed Biotechnol,2007,2007.
Fearon DT, Locksley RM. The instructive role of innate immunity in the acquired immune response. Science,1996,272:50-54.
Fenteany G, Glogauer M. Cytoskeletal remodeling in leukocyte function. Curr Opin Hematol, 2004,11:15-24.
Flannagan RS, Cosio G and Grinstein S. Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nat Rev Microbiol,2009,7:355-366.
Forsyth RB, Candido EPM, Babich SL, et al. Stress protein expression in coho salmon with bacterial kidney disease. J Aquat Anim Health,1997,9(1):18-25.
Freeman BC, Myers MP, Schumacher R, et al. Identification of a regulatory motif in HSP70 that affects ATPase activity, substrate binding and interaction with HDJ-1. EMBO J,1995,14(10): 2281-2292.
Fujiki K, Shin DH, Nakao M,et al. Molecular cloning of carp (Cyprinus carpio) CC chemokine, CXC chemokine receptors, allograft inflammatory factor-1, and natural killer cell enhancing factor by use of suppression subtractive hybridization. Immunogenetics,1999,49:909-914.
Garrity GM. Bergey's Manual of Systematic Bacteriology,2nd edn. Vol.2:The Proteobacteria. New York:Springer-Verlag.2005.
Ghosh J, Buckley K M, Nair SV, et al. Sp185/333:A novel family of genes and proteins involved in the purple sea urchin immune response. Dev Comp Immunol,2010,34:235-245.
Gieselmann V, Hasilik A, von Figura K. Processing of human cathepsin D in lysosomes in vitro. J Biol Chem,1985,260:3215-320.
Gildberg A. Aspartic proteinases in fishes and aquatic invertebrates. Comp Biochem Phys B,1988, 91:425-435.
Glogauer M, Marchal CC, Zhu F, et al. Racl deletion in mouse neutrophils has selective effects on neutrophil functions. J Immunol,2003,170:5652-5657.
Gonzalez-Escalona N, Blackstone GM, DePaola A. Characterization of a Vibrio alginolyticus strain, isolated from Alaskan oysters, carrying a hemolysin gene similar to the thermostable direct hemolysin-related hemolysin gene (trh) of Vibrio parahaemolyticus. Appl Environ Microbial, 2006,72:7925-7929.
Govek EE, Newey SE, Van Aelst L. The role of the Rho GTPases in neuronal development. Genes Dev,2005,19:1-49.
Grace MF, Manning MJ. Histogenesis of the lymphoid organ in rainbow trout. Dev Comp Immunol, 1980,4:255-264.
Gu Y, Filippi MD, Cancelas JA, et al. Hematopoietic cell regulation by Racl and Rac2 guanosine triphosphatases. Science,2003,302:445-449.
Guerrero CA, Bouyssounade D, Zarate S, et al. Heat shock cognate protein 70 is involved in rotavirus cell entry. J Virol,2002,76:4096-4102.
Gupta SK, Lysko PG, Pillarisetti K, et al. Chemokine receptors in human endothelial cells. Functional expression of CXCR4 and its transcriptional regulation by inflammatory cytokines. J Biol Chem,1998,273:4282-4287.
Kneller DG, Cohen FE, Langridge R. Improvements in protein secondary structure prediction by an enhanced neural network. J Mol Biol,1990,214:171-182.
Han JH, Lee JH, Choi YH, et al. Purification, characterization and molecular cloning of Vibrio fluvialis hemolysin. Biochim Biophys Acta,2002,1599:106-114.
Hendrick JP, Hartl F. Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem, 1993,62:349-384.
Henke JM and Bassler BL. Quorum sensing regulates type Ⅲ secretion in Vibrio harveyi and Vibrio parahaemolyticus. J Bacteriol,2004,186:3794-3805.
Hentze M, Hasilik A, von Figura K. Enhanced degradation of cathepsin D synthesized in the presence of the threonine analog beta-hydroxynorvaline. Arch Biochem Biophys,1984,230: 375-382.
Hetman M, Filipkowski R, Domagala W, et al. Elevated cathepsin D expression in kainate-evoked rat brain neurodegeneration. Exp Neurol,1995,136:53-63.
Hightower LE. Heat shock, stress proteins, chaperones and proteotoxicity. Cell,1991,66(2): 191-197.
Hoffman KB, Bi X, Pam JT, et al. Beta amyloid increases cathepsin D levels in hippocampus. Neurosci Lett,1998,250:75-78.
Hoppe AD, Swanson JA. Cdc42, Racl, and Rac2 display distinct patterns of activation during phagocytosis. Mol Biol Cell,2004,15:3509-3519.
Imagawa T, Hashimoto Y, Kon Y. (Cyprinus carpio L)Immunoglobulin containing cells in the head kidney of after bovine serum albumin ininjection. Fish Shellfish Immun,1991,1:173-185.
Isahara K, Ohsawa Y, Kanamori S, et al. Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases. Neuroscience,1999,91:233-249.
Jayaprakash NS, Pai SS, Philip R, et al. Isolation of a pathogenic strain of Vibrio alginolyticus from necrotic larvae of Macrobrachium rosenbergii (de Man). J Fish Dis,2006,29:187-191.
Jeon JH, Kim JT, Kim YJ, et al. Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome. Appl Microbiol Biotechnol,2009,81:865-874.
Jia AR, Zhang X-H. Molecular cloning, characterization and expression analysis of cathepsin D gene from turbot Scophthalmus maximus. Fish Shellfish Immun,2009,26:606-613.
Kazan D, Erarslan A. Identification of catalytically essential amino acid residues of penicillin G acylase obtained from a mutant of Escherichia coli ATCC 11105. Process Biochem,2001,36: 861-867.
Kiang JG, Tsokos GC. Heat shock protein 70 kDa:Molecular biology, biochemistry and physiology. Pharmacol Therapeut,1998,80(2):183-201.
Kim NJ, Sugano Y, Hirai M,et al. Removal of a high load of ammonia gas by a marine bacterium, Vibrio alginolyticus. J Biosci Bioeng,2000,90:410-415.
Kneller DG, Cohen FE, Langridge R. Improvements in protein secondary structure prediction by an enhanced neural network. J Mol Biol,1990,214:171-182.
Kornfeld S, Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol,1989,5:483-525.
Kornfeld S. Lysosomal enzyme targeting. Biochem Soc Trans,1990,18:367-374.
Krebs RA, Feder ME. Natural variation in the expression of the heat-shock protein HSP70 in a population of Drosophila melanogaster and its correlation with tolerance of ecologically relevant thermal stress. Evolution,1997a,51(1):173-179.
Krebs RA, Feder ME. Tissue special variation in HSP70 expression and thermal damage in Drosophila melanogaster larvae. J Exp Biol,1997b,200:2007-2015.
Lee DC, Womble TA, Mason CW, et al.6-Hydroxydopamine induces cystatin C-mediated cysteine protease suppression and cathepsin D activation. Neurochem Int,2007,50:607-618.
Lee JH, Ahn SH, Kim SH, et al. Characterization of Vibrio mimicus phospholipase A (PhlA) and cytotoxicity on fish cell. Biochem Biophys Res Comm,2002,298:269-276.
Li J, Zhou LR, Woo NYS. Invasion route and pathogenic mechanisms of Vibrio alginolyticus to silver sea bream Sparus sarba. J Aquat Anim Health,2003,15:302-313.
Liaudet-Coopman E, Beaujouin M, Derocq D, et al. Cathepsin D:newly discovered functions of a long-standing aspartic protease in cancer and apoptosis. Cancer Lett,2006,237:167-179.
Lindenstrφm T, Secombes CJ, Buchmann K. Expression of immune response genes in rainbow trout skin induced by Gyrodactylus derjavini infections. Vet Immuno Immunop,2004,97:137-148.
Liu L, Fujiki K, Dixon B, et al. Cloning of a novel rainbow trout (Oncorhynchus-mykiss)CC chemokine with a fractalkine-like stalk and a TNF decoy receptor using cDNA fragment containing AU-rich element. Cytokine,2002,17(2):71-81.
Liu PC, Lee KK, Chen SN. Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon. Lett Appl Microbiol,1996,22:413-416.
Liu Y, Chen SL, Meng L, et al. Cloning, characterization and expression analysis of a novel CXC chemokine from turbot(Scophthalmus maximus). Fish Shellfish Immunol,2007a,23:711-720.
Liu Y, Chen SL, Meng L, et al. Cloning, characterization and expression analysis of a CXCL10-like chemokine from turbot (Scophthalmus maximus). Aquaculture,2007b,272: 199-207.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods,2001,25:402-408.
Ma Q, Jones D, Borghesani PR, et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4-and SDF-1-deficient mice. Proc Nat Acad Sci,1998, 95:9448-9453.
Manson FDS, Fletcher TC, Gcbday GW. Localisation of chitinolytic enzymes in blood of turbot, and their possible roles in defence. J Fish Biol,1992,40:919-927.
Mantovani A. The chemokine system:redundancy for robust outputs. Immunol Today,1999,20: 247-290.
Marc Y. Mutiple acute temperature stress affects leucocyte populations and antibody responsed in common carp,Cyrinus carpio L. Fish Shellfish Immunol,2003,15:397-410.
Marie MA, Taylor MD, Blum JS. Endosomal aspartic proteinases are required for invariant-chain processing. P Natl Acad Sci USA,1994,91:2171-2175.
Matos P, Skaug J, Marques B, et al. Small GTPase Rac1:structure, localization, and expression of the human gene. Biochem. Biophys. Res Commun,2000,277:741-751.
Mayer MP. Recruitment of Hsp70 chaperones:a crucial part of viral survival strategies. Rev Physiol Bioch P,2005,153:1-46.
Miyake M, Honda T, Miwatani T. Effects of divalent cations and saccharides on Vibrio metschnikovii cytolysin-induced hemolysis of rabbit erythrocytes. Infect Immun,1989,57: 158-163.
Miyoshi S-I, Sasahara K, Akamatsu S, et al. Purification and characterization of a hemolysin produced by Vibrio mimicus. Infect Immun,1997,65:1830-1835.
Mommsen TP. Salmon spawning migration and muscle protein metabolism:the August Krogh principle at work.. Comp Biochem Phys B,2004,139:383-400.
Montero AB, Austin B. Characterization of extracellular products from an isolate of Vibrio harveyi recovered from diseased post-larval Penaeus vannamei (Bonne). J Fish Dis,1999,22: 377-386.
Montero J, Estepa A, Coll J, et al. Regulation of rainbow trout (Oncorhynchus mykiss) interleukin-8 receptor (IL-8R) gene transcription in response to viral hemorrhagic septicemia virus (VHSV), DNA vaccination and chemokines. Fish Shellfish Immunol,2008,25: 271-280.
Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature,2001,410:50-56.
Murphy PM, Baggiolini M, Charo IF, et al. International Union of Pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev,2000,52:145-176.
Nagasawa T, Tachibana K, Kishimoto T. A novel CXC chemokine PBSF/SDF-1 and its receptor CXCR4:their functions in development, hematopoiesis and HIV infection. Semin Immunol, 1998,10:179-185.
Nielsen H, Brunak S, von Heijne G. Machine learning approaches for the prediction of signal peptides and other protein sorting signals. Protein Eng Des Sel,1999,12:3-9.
Nielsen LB, Nielsen HH. Purification and characterization of cathepsin D from herring muscle (Clupea harengus). Comp Biochem Phys B,2001,128:351-363.
Novoa B, Bowman TV, Zon L, et al. LPS response and tolerance in the zebrafish (Danio rerio). Fish Shellfish Immunol,2009,26(2):326-331.
Ordas MC, Costa MM, Roca FJ, et al. Turbot TNFa gene:Molecular characterization and biological activity of the recombinant protein. Mol Immunol,2007,44:389-400.
Ortuno J, Esteban MA, Meseguer J. Effect of high dietary intake of vitamin C on non-specific immune response of gilthead seadream (Sparus aurata L.).Fish Shellfish Immunol,1999,5: 429-443.
Pablos JL, Amara A, Bouloc A, et al. Stromal-cell derived factor is expressed by dendritic cells and endothelium in human skin. Am J Pathol,1999,155:1577-1586.
Pal S, Guhathakurta B, Sasmal D, et al. Purification and characterization of a hemolysin with phospholipase C activity from Vibrio cholerae 0139. FEMS Microbiol Lett,1997,147: 115-120.
Palmesino E, Moepps B, Gierschik P, et al. Differences in CXCR4-mediated signaling in B cells. Immunobiology,2006,211(5):377-389.
Park KH, Jeong HD. Enhanced resistance against Edwardsiella tarda infection in tilapia(Oreochromis niloticus)by administration of protein bound polysaccharide. Aquaculture, 1996,143:135-143.
Parvathi A, George J, Kumar S. Prevalence and heterogeneity of hemolysin gene vhh among hatchery isolates of Vibrio harveyi in India. Curr Microbiol,2009,59:42-47.
Patel S, Malde K, Lanze'n A, et al. Identification of immune related genes in Atlantic halibut (Hippoglossus hippoglossus L.) following in vivo antigenic and in vitro mitogenic stimulation. Fish Shellfish Immunol,2009,27:729-738.
Pedersen K, Verdonck I, Austin B, et al. Taxonomic evidence that Vibrio carchariae Grimes et al. 1985 is junior synonym of Vibrio harveyi (Johnson and Shunk 1936) Baumann et al.1981. Int J Syst Bacterio,1998,48:749-758.
Press CM, Evensen O. The morphology of the immune system in teleost fishes. Fish Shellfish Immunol,1999,9:309-318.
Qian S, Thai NL, Lu L, et al. Liver transplant tolerance:mechanistic insights from animal models, with particular reference to the mouse. Transplant Rev,1997,11:151-164.
Raida MK, Buchmann K. Innate immune response in rainbow trout (Oncorhynchus mykiss) against primary and secondary infections with Yersinia ruckeri O1. Dev Comp Immunol, 2009,33:35-45.
Reshef L, Ron E, Rosenberg E. Genome analysis of the coral bleaching pathogen Vibrio shiloi Arch Microbiol.2008,190(2):185-94.
Richard V, Dulon D, Hafidi A. Expression of Rho GTPases Rho-A and Racl in the adult and developing gerbil cerebellum. Int. J Dev Neurosci,2008,26:723-732.
Richo G, Conner GE. Structural requirements of procathepsinD activation and maturation. J Biol Chem,1994,269:14806-14812.
Rise ML, Hall J, Rise M, et al. Functional genomic analysis of the response of Atlantic cod (Gadus morhua) spleen to the viral mimic polyriboinosinic polyribocytidylic acid (pIC). Dev Comp Immunol,2008,32(8):916-931.
Roberg K, Kagedal K, Ollinger K. Microinjection of cathepsin D induces caspase-dependent apoptosis in fibroblast. Am J Pathol,2002,161:89-96.
Romano M, Rosanova P, Anteo C, et al. Vertebrate yolk proteins:a review. Mol Reprod pev, 2004,69:109-116.
Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol,2000,18: 217-242.
Salas-Vidal E, Meijer AH, Cheng X, et al. Genomic annotation and expression analysis of the zebrafish Rho small GTPase family during development and bacterial infection. Genomics, 2005,86:25-37.
Sangari FJ, Goodman J, Bermudez LE. Mycobacterium avium enters intestinal epithelial cells through the apical membrane, but not by the basolateral surface, activates small GTPase Rho and, once within epithelial cells, expresses an invasive phenotype. Cell Microbiol,2000,2: 561-568.
Sangrador-Vegas A, Martin S A M, O'Dea PG, et al. Cloning and characterization of the Rainbow trout (Oncorhynchus mykiss) type II interleukin-1 receptor cDNA. Eur J Biochem,2000,267: 7031-7037.
Schmid BC, Rudas M, Rezniczek GA, et al. CXCR4 is expressed in ductal carcinoma in situ of the breast and in atypical ductal hyperplasia. Breast Cancer Res Treat,2004,84:247-250.
Secombes CJ, Zou J, Daniels G, et al. Rainbow trout cytokine and cytokine receptor genes. Immunol Rev,1998,166:333-340.
Selvin J, Lipton AP. Vibrio alginolyticus associated with white spot disease of Penaeus monodon. Dis Aquat Org.2003.57:147-150.
Settleman J. Rho GTPases in development. Prog Mol Subcell Biol,1999,22:201-229.
Sheridan C, Sadaria M, Bhat-Nakshatri P, et al. Negative regulation of MHC class II gene expression by CXCR4. Exp Hematol,2006,34(8):1085-1092.
Shim H, Lau SK, Devi S,et al. Lower expression of CXCR4 in lymph node metastases than in primary breast cancers:Potential regulation by ligand-dependent degradation and HIF-1α. Biochem Biophys Res Commun,2006,346:252-258.
Shima H, Oishic S, Fujii N. Chemokine receptor CXCR4 as a therapeutic target for neuroectodermal tumors. Semin Cancer Biol,2009,19(2):123-134.
Shinoda S, Matsuoka H, Tsuchie T, et al. Purification and characterization of a lecithin-dependent hemolysin from Escherichia coli transformed by a Vibrio parahaemolyticus gene. J Gen Microbiol,1991,137:2705-2711.
Shinoda S. Protein toxins produced by pathogenic vibrios. J Nat Toxins,1999,8:259-269.
Shirai A, Matsuyama A, Yashiroda Y, et al. Global analysis of gel mobility of proteins and its use in target identification. J Biol Chem,2008,283:10745-107452.
Singh V, Somvanshi P, Rathore G, et al. Gene cloning, expression and homology modeling of hemolysin gene from Aeromonas hydrophila. Protein Expres Purif,2009,65:1-7.
Sizemore RG, Miller NW, Cuchens MA, et al. Phylogeny of lymphocyte heterogeneity:The cellular requirements for in vitromitogenic response of channel catfish leukocytes. J Immunol, 1984,133:2920-2924.
Sly LM, Rauh MJ, Kalesnikoff J, et al. LPS-induced upregulation of SHIP is essential for endotoxin tolerance. Immunity,2004,21(2):227-239.
Song YL. Evolution of the lymphoid system Ⅱ.Evidence for immunoglobulin determinants on all rainbow trout lymphcytes and demonstration of mixed leukocyte reaction. Eur J tmmunol, 1977,7:881-887.
Stoline MR. The Status of Multiple Comparisons:Simultaneous Estimation of All Pairwise Comparisons in One-Way ANOVA Designs. Am Stat,1981,35:134-141.
Sultan NAM, Kenoth R, Swamy MJ. Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica. Arch Biochem Biophys,2004,432:212-221.
Sun B, Zhang X-H, Tang X, et al. A Single Residue Change in Vibrio harveyi hemolysin results in the loss of phospholipase and hemolytic activities and pathogenicity for turbot (Scophthalmus maximus). J Bacteriol,2007,189:2575-2579.
Sun CX, Downey GP, Zhu F, et al. Racl is the small GTPase responsible for regulating the neutrophil chemotaxis compass. Blood,2004,104:3758-3765.
Testa J, Daniel LW, Kreger AS. Extracellular phospholipase A2 and lysophospholipase produced by Vibrio vulnificus. Infect Immun,1984,45:458-463.
Thompson JD, Gibson TJ, Plewniak F, et al. The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res, 1997,25:4876-4882.
Tobar N, Caceres M, Santibanez JF, et al. RAC1 activity and intracellular ROS modulate the migratory potential of MCF-7 cells through a NADPH oxidase and NFκB-dependent mechanism. Cancer Lett,2008,267:25-32.
Tong SL, Li H, Miao HZ. The establishment and partial characterization of a continuous fish cell line FG-9307 from the gill of flounder Paralichthys olivaceus. Aquaculture,1997,156: 327-333.
Tsuchida S, Kagi A, Takahashi T. Characterization of cDNA and genomic sequences encoding a canine chemokine receptor, CXCR4 and its ligand CXCL12. Vet Immuno Immunop,2007, 116:219-225.
Vancompernolle K, Herreweghe FV, Pynaert G, et al. Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity. FEBS Lett,1998,438:150-158.
Valencia A, Chardin P, Wittinghofer A, et al. The ras protein family:evolutionary tree and role of conserved amino acids. Biochemistry,1991,30:4637-4648.
Van Noort JM, Van der Drift AC. The selectivity of cathepsin D suggests an involvement of the enzyme in the generation of T-cell epitopes. J Biol Chem,1989,264:14159-14164.
Varani S, Frascaroli G, Homman-Loudiyi M, et al. Human cytomegalovirus inhibits the migration of immature dendritic cells by down-regulating cell-surface CCR1 and CCR5. J Leukoc Biol, 2005,77:219-228.
Vayssier M, Leguerhier F, Fabien JF, et al. Cloning and analysis of a Trichinella briotovi gene encoding a cytoplasmic heat shock protein of 72 kD. Parasitology,1999,119:81-93.
Vega FM and Ridley AJ. SnapShot:Rho Family GTPases. Cell,2007,129.
Wan X, Chen XH. Molecular cloning and expression analysis of a CXC chemokine gene from large yellow croaker Pseudosciaena crocea. Vet Immuno Immunop,2009,127(1-2): 156-161.
Wang CJ, Zhang X-H, Jia AR, et al. Identification of immune-related genes from kidney and spleen of turbot (Scophthalmus maximus) by suppression subtractive hybridization following challenge with Vibrio harveyi. J Fish Dis,2008,31:505-514.
Wang J, Wright MB, Li C, et al. Efficacy of SSH PCR in isolating differentially expressed genes. BMC Genomics,2002,3(1):12.
Wang SX, Zhang X-H, Zhong YB, et al. Genes encoding the Vibrio harveyi haemolysin (VHH)/thermolabile haemolysin (TLH) are widespread in Vibrios. ACTA Microbiol Sinica, 2007,47:874-881.
Wardlaw AC. Practical statistics for experimental biologists. In:John Wiley and Sons. Chichester: New York,1985:29-31.
Watanabe K, Tachibana M, Tanaka S, et al. Heat shock cognate protein 70 contributes to Brucella invasion into trophoblast giant cells that cause infectious abortion. BMC Microbiology,2008, 8:212.
Wei W, Xu H, Wang Q. Identification of differentially expressed genes in large yellow croaker (Pseudosciaena crocea) induced by attenuated live Vibrio anguillarum. Aquaculture,2009, 291:124-129.
Welch WJ. How cells respond to stress. Sci Am,1993,269:56-64.
Wen Y, Shao JZ, Pan XX,et al. Molecular cloning, characterization and expression analysis of QM gene from grass carp(Ctenopharyngodon idellus)homologous to Wilms'tumor suppressor. Comp Biochem Phys,2005,141:356-365.
Wieland H, Goetz F, Neumeister B. Phagosomal acidification is not a prerequisite for intracellular multiplication of Legionella pneumophila in human monocytes. J Infect Dis.2004,189: 1610-1614.
Woods A, Pala D, Kennedy L, et al. Rac1 signaling regulates CTGF/CCN2 gene expression via TGFb/Smad signaling in chondrocytes. Osteoarthr Cartilage,2008,17(3):406-413.
Wu R, Sun L, Lei M, et al. Molecular identification and expression of heat shock cognate 70 (HSC70) in the Pacific white shrimp Litopenaeus vannamei. Mol Biol,2008,42:265-274.
Xu H, Yan F, Deng X, et al. The interaction of white spot syndrome virus envelope protein VP28 with shrimp Hsc70 is specific and ATP-dependent. Fish Shellfish Immunol,2009,26: 414-421.
Yao CL, Wu CG, Xiang JH, et al. The lysosome and lysozyme response in Chinese shrimp Fenneropenaeus chinensis to Vibrio anguillarum and laminarin stimulation. J Exp Mar Biol Ecol,2008,363:124-129.
Yasuko S, Chuzo I, Yukiko I, et al.71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human T-cell lymphotropic virus type 1. J Virol, 1998,72:535-541.
Yong QU. Lysosomal storage disorders. J Peking university (health sciences),2006,38:100-102.
Yost HJ, Lindquist S. RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell,1986,45(2):185-193.
Yost HJ, Lindquist S. Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae. Mol Cell Biol,1991,11(2):1062-1068.
Zhang YB, Chen XP, Tao LY, et al. Expression of Cathepsin-B and-D in Rat's Brain after Traumatic Brain Injury. J Foren Med,2006,22:404-406.
Zhang X-H, Austin B. Pathogenicity of Vibrio harveyi to salmonids. J Fish Dis,2000,23:93-102.
Zhang X-H, Austin B. Haemolysins in Vibrio species. J Appl Microbiol,2005,98:1011-1019.
Zhang X-H, Meaden PG, Austin B. Duplication of hemolysin genes in a virulent isolate of Vibrio harveyi. Appl Environ Microbiol,2001,67:3161-3167.
Zhao XX, Carnevale KA, Cathcart MK. Human monocytes use Racl, not Rac2, in the NADPH oxidase complex. J Biol Chem,2003,278:40788-40792.
Zheng WB, Tian C, Chen XH. Molecular characterization of goose-type lysozyme homologue of large yellow croaker and its involvement in immune response induced by trivalent bacterial vaccine as an acute phase protein. Immunol Lett,2007,113:107-116.
Zhong QW, Zhang QQ, Wang ZG, et al. Expression profiling and validation of potential reference genes during paralichthys olivaceus embryogenesis. Mar Biotechnol,2008,10:310-318.
Zhong Y, Zhang X-H, Chen J, et al. Overexpression, purification, characterization and pathogenitiy of Vibrio harveyi haemolysin VHH. Infect Immun,2006,74:6001-6005.
Zhou H, Wang H. Chemical modification of proteins. Tsinghua University Press, Beijing,1988: 19-43.
Zitzer AI, Palmer WM, Bhakdi S. Characterization of Vibrio cholera El Tor cytolysin as an oligomerizing pore-forming toxin. Med Microbiol Immunol,1995,184:37-44.