Cloning And Expression Analysis Of Three Immune Genes From Paralichthys Olivaceus

Chemokines are small, secreted cytokine peptides(6-14 k Da),which were initially found to have the ability to recruit a wide range of immune cells to sites of infection and disease in mammals. Based on the arrangement of the first two cysteine residues of the amino acid sequences, chemokines were divided into four groups: CXC, CC, CX3 C and C subfamilies. The first two cysteines are separated with one intervening amino acid in CXC chemokines. Chemokine receptors are composed of seven transmembrane domain–containing G protein–coupled receptors(GPCRs). Chemokine receptors and their ligands have been identified as major factors initiating and controlling the migratory patterns of all immune cells, and are critical for the fun ctions of the innate and adaptive immune responses. Depending on the combination of chemokines, chemokine receptors can be divided into CXCR1- CXCR5(combined with CXC), CCR1- CCR9(combined with CC), XCR1(combined with C), CX3 CR(combined with CX3C).Paralichthys olivaceus is an important economic marine fish species in the world due to its rapidgrowth and good taste, widely breeding in China, South Korea and Japan and other Asian countries. However, with the continuous development of high-density factory farming and environmental deterioration in the breeding, the aquacultural fish diseases have occurred frequently,which have limited profitability and development of aquaculture. The use of antibiotics has raised concerns of antibiotic residues in fish, polluting environment and antibiotic resistance development, although it has partially solved the problem. Therefore, it is very necessary to improve disease resistance using molecular technique. Cloning and characterization of fish chemokines could provide a way to modify the fish immune system and improve their disease resistance in aquaculture.1 Cloning, characterization and expression analysis of a CXCL9 chemokine from Japanese flounder(Paralichthys olivaceus)In this study, we cloned the CXCL9 genes of the Paralichthys olivaceus. The full-length CXCL9 c DNA is 910 bp, contains a 56 bp 5’Untranslated Region, an open reading frame of 408 nucleotides encoding 135 amino acid residues and a 3’ Untranslated Region, respectively. The predicted cleavage of the signal sequence to the putative mature peptide is between glycine28 and threonine acid29.The 3’ Untranslated Region containsa single typical polyadenylation signal(AATAAA).Four conserve cysteine residues at 35, 37, 60 and 77, and the first two cysteines are separated with one leucine acid which is exactly consistent with the feature of CXC chemokine. Amino acid sequence analysis revealed that Paralichthys olivaceus CXCL9 shared high homology with the CXC gene of Scophthalmus maximus, Anoplopoma fimbriaand and Sparus aurata, respectively 82%、74% and 68%. RT-PCR demonstrated that Paralichthys olivaceus CXCL9 chemokine was expressed highly in liver, and higher in spleen, skin and gill. The highest expression levels of the Paralichthys olivaceus CXCL9 chemokine were detected at 6h in liver after challenge with V. anguillarum and E. tarda and decreased gradually at lower levels. These results indicated that the Paralichthys olivaceus CXCL9 chemokine played an important role against pathogens infection in Paralichthys olivaceus immune response.2 Molecular cloning, structure and expressional profiles of two novel single-exon genes( Po CCR6 A and Po CCR6B) in the Japanese flounder( Paralichthys olivaceus)CCR6 is an important binding receptor of CCL20 and beta-defensins, and has multiple functions in the innate and acquired immune responses. In this study, we cloned the Po CCR6 A and Po CCR6 B genes of the Japanese flounder and studied the gene structure and expression patterns of these two genes in bacterial infection. The full-length Po CCR6 A c DNA is 1415 bp and the open reading frame(ORF) is 1113 bp, encoding a 370-amino-acid peptide. The full-length Po CCR6 B c DNA is 2193 bp and the ORF is 1029 bp, encoding a 363-amino-acid peptide. The structures of Po CCR6 A and Po CCR6 B indicate that they are single-exon genes. The predicted proteins encoded by Po CCR6 A and Po CCR6 B have the typical G-protein-coupled receptor(GPCR) family signature of seven transmembrane domains and several conserved structural features. A tissue distribution analysis showed that Po CCR6 A is predominately expressed in the intestine, gill, and blood, and Po CCR6 B in the gill, spleen, and liver. The expression patterns of the two chemokine receptors were analyzed during bacterial infection. In spleen and kidney, the expression of Po CCR6 A was significantly upregulated at 24 h after infection, whereas the expression of Po CCR6 B was steady at these time points. While in intestine, both of them were upregulated at 6 h to 12 h after infection, and in gill the expression levels of them were upregulated at 24 h. The patterns of expression suggested that Po CCR6 A and Po CCR6 B play an important role in the immune response of the Japanese flounder, especially in the mucosal tissues.