Molecular characterization and expression analysis during embryo development of CD4–1 homologue in large yellow croakerLarimichthys crocea

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CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4–1 homologue, LycCD4–1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4–1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4–1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4–1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4–1 has 27.9–58.4% identity to other teleost fish CD4–1 molecules, and 16.4–20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4–1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4–1 form a cluster with CD4–1 molecules in other fish species. The LycCD4–1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4–1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4–1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4–1 and tracing origin of CD4–1+ cell precursors in fish.

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