Characterization Of CD3ε,CD3γ/δ And CD3ζ In Large Yellow Croaker (Larimichthys Crocea) And Development Of Polyclonal Antibody Against CD3ε

In this study,CD3γ/δA,CD3γ/δB,CD3εand CD3ζwere cloned and identified in large yellow croaker（Larimichthys crocea）,their open reading frame（ORF）sequences were 522,573,555 and 456 bp in length,encoding 173,190,184 and 151 amino acids,respectively.The deduced LcCD3γ/δA,LcCD3γ/δB and LcCD3εproteins have two conserved cysteines and a CXXCXE motif in extracellular region,and an immunoreceptor tyrosine-based activation motif(ITAM,YXXL/I-X_6-8-YXXL/I)related to signal transduction in the intracellular region.LcCD3εhas a conserved aspartic acid residue in transmembrane domain,while LcCD3γ/δA and LcCD3γ/δB have an asparagine.LcCD3ζdiffers from other LcCD3 subunits,with an extreme short extracellular sequence（8 aa）and three potential ITAMs in intracellular region.Multiple sequence alignment showed that LcCD3εand LcCD3γ/δhave the highest identity to mandarin fish CD3γ/δand CD3εrespectively.LcCD3ζhave the highest identity to blood-red snapper CD3ζ.All LcCD3 subunits mRNA were constitutively expressed in all examined tissues and immune-related cells.The mRNA expression level of LcCD3γ/δA and LcCD3γ/δB were high in intestines,blood,gills and spleen,and that of LcCD3εand LcCD3ζwere high in gills and spleen.The mRNA expression levels of all LcCD3 subunits were highest in primary head kidney leukocytes（PKLs）.Upon Vibrio.Alginolyticu induction,mRNA expression levels of LcCD3 subunits were up-regulated in the spleen and head kidney,climbing to the peak at 24 h after induction.The mRNA expression of LcCD3 subunits were up-regulated in PKLs,reaching the peak at 8 h with PHA or Con-A stimulation.These results indicated that CD3 may participate in anti-microbial response and activation of T cells.Moreover,Recombinant LcCD3εprotein was produced in Escherichia coli and used for anti-r LcCD3εpolyclonal antibody production.The protein level of LcCD3εand transcriptional levels of LcCD3εand cytokines（Lc IL-2,Lc IFN-γ）were up-regulated in spleen stimulated with recombinant Dihydrolipoyl dehydrogenase（DLD）protein.In conclusion,LcCD3γ/δA,LcCD3γ/δB,LcCD3εand LcCD3ζwere cloned and identified in this paper,and the results of our studies implied that CD3 may be involved in resistance to bacterial host immune response,and play a role in T cell activation.