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| Cloning and Expression Analysis of CD63 Gene in Lateolabrax japonicus |
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Abstract Abstract The cluster of differentiation 63 (CD63) belongs to the tetraspanin superfamily, which plays an important role in immuno-physiological functions including cell adhesion, signal transduction and immune cell activation. In recent years, there are serious disease problems in the aquaculture of Lateolabrax japonicus, in order to strengthen the basic research for fish diseases and immune. In this study, CD63 cDNA sequence of Lateolabrax japonicus was cloned using rapid-amplification of cDNA ends (RACE) method. The protein molecular weight, isoelectric point and structure prediction using online software. To expression analysis of CD63 using qRT-PCR. The experimental results showed that the amplified sequence was 1 219 bp, including 114 bp 5'-UTR, 382 bp 3'-UTR and 723 bp ORF which encoding 240 amino acids. It was predicted that the molecular weight of protein was 26.26 kD and the isoelectric point is 8.43. The results of online software analysis showed that the protein was a 4 transmembrane protein, including 4 transmembrane regions (13~35, 50~72, 85~107 and 207~229 aa), three intracellular domains (1~12, 73~84 and 230~240 aa) and 2 extracellular loops (36~49, 108~206 aa). A conserved Cys-Cys-Gly motif and 3 potential N-glycosylation sites (Asn128, Asn150 and Asn172) were found in the large extracellular loop region. The carboxy terminus of the protein had a tyrosine lysosome target sequence (GYEVM, Gly-Tyr-Glu-Val-Met). Bioinformatics analysis indicated that Lateolabrax japonicus CD63 peptide share 61%~84% similarity with other fish counterparts. Quantitative real-time PCR showed that the mRNA of CD63 was expressed in all tissues examined, including liver, spleen, head kidney, intestines, gill, fat, muscle, brain and blood. The higher expression of CD63 was detected in the liver, muscle and head kidney. The expression level of CD63 in head kidney didn't show any difference at 3 h after intraperitoneal injection with Vibrio harveyi, but up-regulated significantly at 6 h (P＜0.05) and down-regulation at 12 h. In vitro experiment, the mRNA expression of CD63 showed significantly up-regulation in head kidney macrophages after stimulation with LPS 12 h (P＜0.05). The CD63 expression were also significantly up-regulated in macrophages at 24 h after stimulating by Poly I:C (P＜0.05). The results indicated that CD63 may play important roles in pathogen invasion and inflammation reaction. This provides the theoretical basis for the further study of fish immune response molecules regulatory networks.
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Received: 11 September 2017
Published: 02 May 2018
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