Immunopurification of T-cells from sea bass Dicentrarchus labrax (L.).
The monoclonal antibody DLT15, specific for thymocytes and peripheral T-cells of the teleost fish Dicentrarchus 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 TcR beta chain, a 203 bp product was amplified. When sequenced, the cDNA was found to show 60% nucleotide identity to the trout TcRV beta 3. 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.
Scapigliati
G
G
Dipartimento di Scienze Ambientali, Università della Tuscia, Viterbo, Italy. scapigg@unitus.it
Romano
N
N
Abelli
L
L
Meloni
S
S
Ficca
A G
AG
Buonocore
F
F
Bird
S
S
Secombes
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001492:Bass / Q000276:immunology*; D002469:Cell Separation / Q000662:veterinary; D004064:Digestive System / Q000166:cytology* / Q000276:immunology; D005434:Flow Cytometry / Q000662:veterinary; D018928:Immunity, Mucosal; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D018189:Immunomagnetic Separation / Q000662:veterinary; D008221:Lymphoid Tissue / Q000166:cytology; D008969:Molecular Sequence Data; D010802:Phylogeny; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D013601:T-Lymphocytes / Q000276:immunology*
D007135:Immunoglobulin Variable Region; D016693:Receptors, Antigen, T-Cell, alpha-beta