| 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. |
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