Molecular cloning and characterization of two types of CD8alpha from ginbuna crucian carp, Carassius auratus langsdorfii

We cloned and sequenced full-length cDNAs for two types of CD8alpha from the S3n strain of ginbuna crucian carp (Carassius auratus langsdorfii) and quantified the expression of CD8alpha genes after sensitization by scale grafting, employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. RT-PCR yielded four different fragments of CD8alpha homologue from the S3n strain of ginbuna and these sequences were classified into two groups. The two types of ginbuna CD8alpha (gbCD8alpha) were also found in other strains of triploid ginbuna and goldfish, which are a subspecies of ginbuna. The gbCD8alpha chains consisted of a signal peptide, Ig superfamily (IgSf) V-like domain, hinge, transmembrane domain, and cytoplasmic domain similar to other known CD8alpha. Phylogenetic analysis indicated that both types of gbCD8alpha are closely related to CD8alpha from other vertebrates. Expression of both types of gbCD8alpha mRNA was detected in the gill, thymus, head kidney, posterior kidney, spleen, intestine and peripheral blood leucocytes. In addition, quantitative real-time PCR analysis demonstrated that copy numbers of both gbCD8alpha gene products in kidney cells increased significantly following grafting with allogeneic but not isogeneic scales, and that regulation of expression correlated with that of TCRbeta. Expression of both gbCD8alpha genes after second scale allografting was elevated compared to that after the first set of grafting. These results suggest that expression analysis of these two gbCD8alpha sequences provides a useful tool to address the involvement of cytotoxic T-lymphocytes during the cell-meditated immune response in fish.