الفهرس 
GENERAL INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 115 |  |  |
| | | from | 115 | | |
Abstract
Comprehensive identification of immune-related genes in fish by genome and transcriptome analysis is a promising approach to full understanding of their immune system needed for its application to disease transcriptome (EST) analysis of the gill and intestine, which are considered to be important interfaces for outer environment and potential sites of microbial infections, using the common carp (Cyprinus carpio) stimulated with fungal β-glucan and pathogen bacteria Edwardsiella tarda. Sequence analyses of 2148 EST clones from both organs resulted in identification of 195 putative immune –related genes, including a novel IL-8-like chemokine, designated CaIL-8, found in the gill ESTs. The CaIL-8 transcript encodes 99 amino acids containing a typical CXC motif of this chemokine family but lacks an ELR-motif, as in most teleost IL-8-like chemokines. Phylogenetic tree suggested a closer relationship of call-8 with mammalian IL-8 orthologues than with other teleost CXC-chemokines reported to be IL-8-like. In normal unstimulated carp, Call-8 mRNA was detected by RT-PCR in immune organs such as kidney, spleen, gill and peripheral blood leukocytes (PBL), in contrast to previously reported carpIL-8-like chemokine CXCa, which shows ubiquitous basal expression. Upon stimulation in vitro with lipopolysaccharide (LPS) or poly (I:C) , CaIL-8 and CXCa responded differently in the gill , showing significant down-regulation of CaIL-8 but not CXCa , while both were up-regulated by LPS in the spleen , head kidney and hepatopancreas. Recombinant protein of CaIL-8 (rCaIL -8) was produced in a bacterial system as inclusion body, and then successfully recovered as soluble from through an on-column refolding technique. The rCaIL-8 protein, over concentrations of 5 to 200 ng/ml, showed a significant chemoattractive activity to neutrophils, lymphocytes and macrophages at similar efficiencies, collected from carp head kidney. Polyclonal anti-CaIL-8 antibody was raised in rabbits as a tool to characterize natural CaIL-8 from both PBL cell lysate and their culture supernatant, after incubation with or without various leukocyte stimulants. Overall, this study provides a line of molecular and biochemical evidence for presence of two divergent lineages of IL-8-like CXC-chemokines in teleosts, and for functional activity of CaIL-8 in carp leukocytes chemotaxis. In particular, the presence of natural IL-8-like molecule in tissues was proven by this study for the first time in fish, providing a concrete starting point for further research development about immunomodulating functions of the CXC-chemokines in fish.