| Serum of large yellow croaker was collected at 4h, 1d, 2d, 4d, 8d, 12d, 16d after intraperitoneal injection with Vibrio parahaemolyticus(experimental group)or 0.9% NaCl solution (control group), and the response of selected innate immune parameters (nitric oxides synthase, lysozyme and tyrosinase) was investigated. The lysozyme activities in the serum of large yellow croaker in the experimental group were significantly higher than that of the control group at 2d and 12d after injection, while at 16d after injection, it was significantly lower than that of the control group. The tyrosinase activities in the serum of large yellow croaker in the experimental group were significantly higher than that of the control group at 4d and 16d, and at 8d after injection it was much significantly higher than that of the control group. Nitric oxides synthase activities did not show statistical difference between experimental and control group during the exposure. These results indicate that lysosyme and tyrosinase play an important role in immune defence of V. parahaemolyticus in large yellow croaker.Base on the above results, RT-PCR and real time PCR were used to clone and analyse the four genes of TNFα, Mx, IgL and IgH from large yellow croaker. The main results are reported as follow: (1) The full length cDNA of TNFαcontained a 5'UTR of 68 bp, followed by an ORF of 759 bp, and a 3'UTR of 512bp. The ORF was capable of encoding 241 amino acids with an estimated molecular mass of 28 kDa. The deduced peptide had a transmembrane domain and a TNFαfamily signature. Large yellow croaker TNFαshares relatively high similarity with both teleost and mammalian TNFαby multiple sequence alignments. Phylogenetic analysis showed that the teleost TNFαwere located independently in a different branch compared with mammalian TNFα. Real time PCR analyses demonstrated that the expression of TNFαin head kidney of large yellow croaker injected with V. parahaemolyticus were much significantly higher than that of the control group at 2d and 4d; in blood, at 8d after injection it was significantly higher than that of the control group, while at 4d after injection it was much significantly higher than that of the control group. (2) The Mx full length cDNA of 2209 bp contained an ORF of 1890bp that coded for a protein of 629 amino acids with an estimated molecular mass of 72 kDa. Within the coding sequence, characteristic features of Mx proteins were found, such as a GTP-binding motif, the signature of the dynamin family (LPRGSGIVTR), and a sequence that coded for a leucine zipper at the C-terminal region of the protein. Intraperitoneal challenge of large yellow croaker with V. parahaemolyticus significantly up-regulated Mx expression in head kidney and blood at 2d, and also peaked at 2d. (3) The 977bp full length cDNA of IgL and 1621bp fragment cDNA of IgH have been cloned. IgL encoding comprising its variable (VL) and constant (CL) regions, which were conserved with their putative domains; and its exchanges were present mainly within CDRs. The phylogenetic analyses also demonstrated that large yellow croaker and other piscine IgL cluster as a separate branch out of the mammalian branches. Moreover, real time PCR revealed that the expression of IgL and IgH in head kidney large yellow croaker injected with V. parahaemolyticus were significantly higher than that of the control group at 8d and 12d, respectively.Annealing control primer (ACP) system is a novel method that is based on DDRT-PCR, which can be used to identify differentially expressed genes. The primer comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3′end target core sequence and the 5′end non-target universal sequence, resulting in a dramatic improvement of annealing specificity. It has the key features of higher reproducibility, low false positives, speed and cost-effectiveness and a wide range of PCR products. We have used this method with some modification to clone a 1360bp fragment of fibrinogen beta chain gene which response to a pathogen challenge in large yellow croaker.
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