Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.)

Abstract

The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fishDicentrarchus labrax (sea bass), was used to purify immunoreactive cells from blood and gut-associated lymphoid tissue. The purification was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of the isolated cells was estimated by cytofluorimetric analysis. Following a single step, the percentage of DLT15-purified cells was 88±10% for gut-associated lymphoid tissue and 79±18% for blood leucocytes. DLT15-purified cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT-PCR experiments using as primers degenerate oligonucleotides corresponding to the peptide sequence MYWY and VYFCA of the trout TcRβ chain, a 203bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRVβ3. By 3′-RACE the cDNA was elongated to obtain the TcR constant region, with high similarity to other fish TcR sequences. These results strongly suggest that cells recognised by DLT15 are putative T lymphocytes.