【作者】 贾爱荣；

【导师】 张晓华；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2010， 博士

【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。更多还原

【Abstract】 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.更多还原