Molecular And Expression Characterizations Of Some Disease-resistant Chemokines In Large Yellow Croaker,Larimichthys Crocea

Large yellow croaker, Larimichthys crocea, one of the largest amount of mari-cultured fish species in China, is suffering very severe natural resources exhausted and serious disease caused by viral, bacterial and parasitic infection in recent years, which results in huge economic losses in its culture industry. However, the immune response mechanism of it is still little known.In the present study, the full length cDNA of IL-6, IL-8, IL-12p35, IL-12p40 and type I IFN were cloned from large yellow croaker by RT-PCR and RACE-PCR, and then the genomic structure of these genes were described. In addition, the tissue expression and temporal expression profiles of these genes in spleen, head-kidney and liver after the stimulation with LPS(lipopolysaccharide), poly I:C and pathogenic bacteria Vibrio parahemolyticus were described. We also reported the recombinant expression of IL-8 gene and investigated the some SNPs of in IL-8 genomic sequence. The results are as follows:(1) The full-length cDNA of LcIL-6 was 1066 bp, containing a 71 bp 5’ untranslated region(UTR), a 317 bp 3’ UTR and a 678 bp open reading frame(ORF) which encoded 225 amino acids, containing an IL-6 domain. The full-length genomic sequence of LcIL-8 was composed of 2126 nucleotides, including five exons and four introns. Quantitative real-time PCR(qRT-PCR) analysis indicated a broad expression of LcIL-6 in most detected tissues, with the most predominant expression in stomach and the weakest expression in gill. After injection with LPS and V. parahemolyticus, LcIL-6 expression levels showed up-regulation in head-kidney, liver and spleen. Poly I:C could make LcIL-6 transcripts show up-regulation in the liver and spleen.(2) The full-length cDNA of LcIL-8 was 931 bp, containing a 118 bp 5’ UTR, a 528bp3’-UTR and a 285 bp ORF which encoded 94 amino acids, containing a small cytokine(SCY)domain. The full-length genomic sequence of LcIL-8 was composed of 1930 nucleotides,including four exons and three introns. qRT-PCR analysis indicated a broad expression of LcIL-8 in most detected tissues, with the most predominant expression in liver and the weakest expression in spleen. After injection with LPS, V. parahemolyticus and poly I:C, LcIL-8expression levels showed up-regulation in head-kidney and spleen. However, LcIL-8 transcriptsshowed down-regulation in the liver after all the three stimulants injection. The expression conditions of IL-8 gene recombinant expression is that using IPTG(0.5mM) to induce for 6 h at30℃. Furthermore, 5 single nucleotide polymorphisms(SNPs) were identified in Lc IL-8 gene.(3) The full-length cDNA of LcIL-12p35 was 972 bp, containing a 35 bp 5’ UTR, a 337 bp3’ UTR and a 600 bp ORF which encoded 199 amino acids, containing an IL-12 domain. The full-length cDNA of LcIL-12p40 was 1276 bp, containing a 48 bp 5’ UTR, a 160 bp 3’ UTR and a 1068 bp ORF which encoded 355 amino acids, containing an IL-12p40_C domain. The full-length genomic sequence of LcIL-12p35 was composed of 1745 nucleotides, including seven exons and six introns. The full-length genomic sequence of LcIL-12p40 was composed of3106 nucleotides, including eight exons and seven introns. qRT-PCR analysis indicated a broad expression of LcIL-12p35 and p40 in most detected tissues, with the same most predominant expression in stomach and the same weakest expression in gill. After injection with LPS,LcIL-12p35 expression levels showed up-regulation in liver. Poly I:C could make LcIL-12p35 transcripts show up-regulation in the head-kidney, liver and spleen. After injection with V.parahemolyticus, LcIL-12p35 expression levels showed up-regulation in head-kidney and liver.While, in spleen, LPS and poly I:C could make the LcIL-12p40 expression levels showed up-regulation. The three stimulations induce the LcIL-12p40 expression levels up-regulation in head-kidney and down-regulation in liver.(4) The full-length c DNA of LcIFN I was 878 bp, containing a 62 bp 5’ UTR, a 258 bp3’UTR and a 558 bp ORF which encoded 185 amino acids, containing an IFabd(Interferon alpha, beta and delta) domain. qRT-PCR analysis indicated a broad expression of LcIFN I in most detected tissues, with the most predominant expression in blood cells and the weakest expression in muscle. After injection with poly I:C, LcIFN I expression levels showed up-regulation in head-kidney, liver and spleen; After injection with LPS, Lc IFN I expression levels showed up-regulation in liver and spleen; LcIFN I transcripts showed up-regulation in the liver and head-kidney after V. parahemolyticus injection.