Cloning, Characterization And Expression Analysis Of 5 Chemokines From Turbot (Scophthalmus Maximus)

Chemokines are a large family of cytokines involved in recruitment, activation and adhesion of a variety of leukocytes to inflammatory foci, as well as in the organization and maintenance of lymphoid organ architecture and in normal developmental processes. They are important components of innate immune system. Most chemokines contain four conserved cysteine residues. Based on the arrangement of these conserved cyssteine residues, chemokines are divided into four subfamilies: CXC(α),CC(β),C(γ) and CX3C(δ). The CXC and CC are the two major subfamilies. Five chemokines were screened from a spleen cDNA library and a headkidney cDNA library of turbot (Scophthalmus maximus). Among the five chemokines, two (S457 and S950) are CXC chemokines and three (K1063, SC70 and KC70) are CC chemokines.Partial cDNA sequences were obtained from the two libraries. Unknown 5' and 3' sequences were cloned with specific primers using rapid amplification of cDNA ends (RACE) in order to obtain the cDNA of full length. The genomic sequences were also cloned using PCR with the specific primers, which were designed based on their open reading frames (ORF). The five chemokine genes have four exons and three introns except SC70, which has three exons and two introns. Their amino acid sequences all have four conserved cysteine residues. The conserved domains for chemokine were determined by BlastP and the signal peptides were analysed by SignalP program.An NJ phylogenetic tree was constructed between fish CXC chemokines and other well-known CXC chemokines. The NJ tree contains four clades, which include fish CXC chemokines. CXC chemokine S950 is more closer to CXCL10. but it is difficult to define S457, which is close to CXCL9, CXCL10 and CXCL11. An NJ phylogenetic tree was also constructed between fish CC chemokines and other well-known CC chemokines. In the tree, there are five clades including both fish and mammalian CC chemokines. There is also a clades in which all are fish CC chemokines. SC70 was found to be close to CCL25, and KC70 close to CCL19. It is difficult to identify K1063.Semi-quantitative analysis using RT-PCR was conducted in order to determine the expressed level of the five chemokine in normal turbot tissues, different embryonic periods, turbot embryonic cells (TEC) and individuals infected by Vibrio anguillarum. The results was listed as followes:(1) In 11 tissues of normal turbot, S457 was expressed strongly in immune-related organs, especially spleen and kidney, but faintly in headkidney. S950 was not expressed in all 11 tissues. K1063 was expressed strongly in liver, spleen, headkidney and kidney. The expression of KC70 was detected in the tissues exclude brain and skin. The expression of SC70 was detected in all tissues.(2) During different embryonic periods, S457 was expressed faintly after somites stage and then changes not significantly. SC70 was expressed after pre-hatching stage, and strongly expressed in larvae. KC70 was strongly expressed after gastrula stage. S950 and K1063 was not expressed during embryonic period.(3) In TECs infected by Vibrio anguillarum, only the strong expression of S457 was detected at 6h after infection, and the level of expression decreased after 12h.(4) In the three tissues(liver, spleen and headkidney) of turbot infected by Vibrio anguillarum , The expressed level of S457 and S950 increases and then decreases gradually. K1063 and SC70 have the same trend as S457 and S950. Vibrio anguillarum has no effects on the expression of KC70.