Characterization and expression of a CD40 homolog gene in Japanese flounder Paralichthys olivaceus

A CD40 homolog cDNA encoding 300 amino acid residues was isolated from a Japanese flounder leukocyte cDNA library. The amino acid sequence identities of Japanese flounder CD40 and previously reported CD40s of cow, human, mouse, and chicken range from 32 to 35%. The positions of cysteine residues, CD40 ligand binding amino acid residues, and four cysteine-rich domains are well conserved in Japanese flounder CD40. The Japanese flounder CD40 gene is composed of nine exons and eight intervening introns spread over 6 kb. The nucleotide sequence of the 5′-flanking region of this gene revealed the presence of several regulatory regions, including a TATA-like box, AP-1-, CEBPB-, IRF-1-, LYF-1-, NF-κBp-, SP-1-, and Stat-1-like motifs. In healthy fish, reverse transcriptase–polymerase chain reaction (RT-PCR) detected constitutive expression of CD40 in all tested tissues (leukocytes, kidney, spleen, liver, intestine, brain, gill, and skin). The mRNA of CD40 was predominantly expressed in several tissues that contained lymphocytes (leukocytes, kidney, spleen, intestine, and gill). Expression of the Japanese flounder CD40 molecule was induced in peripheral blood leukocytes from 1 to 6 h following Con A (50 μg/ml)/phorbol myristate acetate (PMA) (0.35 μg/ml) stimulation, with a peak at 1 h after stimulation, and increased at 1, 3, and 6 h after induction by lipopolysaccharide (LPS) (500 μg/ml) compared with the control.