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Cloning and expression analysis of the co-receptor CD8α in sea bream (Sparus aurata L.)

2006; Elsevier BV; Volume: 256; Issue: 1-4 Linguagem: Inglês

10.1016/j.aquaculture.2006.02.013

1873-5622

Elisa Randelli, Alessio Foglietta, Massimo Mazzini, Giuseppe Scapigliati, Francesco Buonocore,

T-cell and B-cell Immunology

Abstract In this paper we describe the molecular cloning and tissue expression of the CD8α from sea bream (Sparus aurata L.). Primers derived from conserved regions of the CD8α extracellular Ig superfamily (IgSf) V domain were used on sea bream thymus cDNA to search for this molecule. A product of 222 bp, the expected size, corresponding to CD8α was obtained and employed to design specific primers for 3′ and 5′ RACE-PCR to complete the sequence. The sea bream CD8α full length cDNA is comprised of 886 bp and is translated in one reading frame giving a deduced protein of 225 amino acids, with a predicted 21 amino acids signal peptide, a 34 bp 5′-UTR and a 177 bp 3′-UTR. A multiple alignment of CD8α from sea bream with other known fish CD8α sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8αs. However, the motif CXCP that is thought to be responsible for p56lck binding in mammals is missing in the sea bream sequence as well as in all fish sequences, where is replaced by CPHH, with the first cysteine (sea bream C-179) conserved from fish to mammals. Phylogenetic analysis conducted employing deduced amino acid sequences showed that sea bream CD8α grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8α sequences. By RT-PCR analysis on unstimulated fish the thymus was the tissue showing the highest CD8α expression, whereas lower mRNA levels were found in spleen, gut, gills, head kidney and liver. This study will help towards the development of new tools for the characterisation of specific T-cell mediated immunity in sea bream, a fundamental species for the Mediterranean fish farms.