Sequencing and expression analysis of CD3γ/δ and CD3εchains in mandarin fish, Siniperca chuatsi

摘要

The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish,Siniperca chuatsi,were determined.As in other vertebrate CD3 molecules,the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ε contained conserved residues and motifs,such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs.However,mandarin fish CD3γ/δ and CD3ε showed some differences to their mammalian counterparts,specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ.Additionally,while an N-glycosylation site was present in CD3ε,the site was not observed in CD3γ/δ.The CD3γ/δ and CD3ε subunit sequences contain six and five exons,respectively,consistent with homologues from Atlantic salmon,Salmo salar.Phylogenetic analysis also revealed that CD3γ/δ and CD3ε in mandarin fish are closely related to their counterparts in Acanthopterygian fish.Real-time PCR showed CD3γ/δ and CD3ε were expressed mainly in the thymus and spleen in normal healthy fish and,to a lesser extent,in mucosal-associated lymphoid tissues,such as the intestine and gills.When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin,concanavalin,and polyriboinosinic polyribocytidylic acid,mRNA expression levels of CD3γ/δ and CD3ε were significantly elevated within 12 h of treatment.This indicated the presence of T lymphocytes in the head kidney of teleost fish,and also the recognition of mitogens by the lymphocytes.Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ε mRNA,indicating that CD3γ/δ and CD3ε lymphocytes are involved in the immune response of this species.