Characterization and expression of Cd8 molecules in mandarin fish Siniperca chuatsi

The full-length complementary DNA (cDNA) sequences encoding cd8α and cd8β molecules were sequenced and characterized from mandarin fish Siniperca chuatsi. Conserved motifs and residues were found to be present in derived peptides of the Cd8 molecules. For example, WXR motif, DXGXYXC motif, and four cysteine residues were present in the extracellular region of the Cd8 protein. Threonine, serine and proline residues involved in multiple O-linked glycosylation events were located in the membrane proximal hinge region. The common CPH motif in the cytoplasmic tail was detected similar to other teleost Cd8 molecules. Different from those in mammals, S. chuatsi Cd8 sequences have many extra cysteine residues (C149 in Cd8α sequence and C46, C51 and C158 in Cd8β sequence), which also exist in other teleost Cd8 molecules. Real-time polymerase chain reaction (RT-PCR) and Western blot analyses revealed that the thymus had the highest expression of cd8 messenger (m)RNA and protein. After stimulated with phytohaemagglutinin, polyriboinsine-polyribocyaidylic acid and concanavalin A (ConA), the expression level of cd8 mRNA increased significantly in head-kidney lymphocytes at 4 and 8 h, but decreased to normal level at 12 h. Similarly, stimulation with ConA in vivo also led to an increase in the cd8 mRNA level in the spleen. Immunohistochemistry analysis demonstrated that Cd8α-positive cells can be detected in the thymus, spleen and intestine by using polyclonal anti-Cd8α antibody.