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

Fish Shellfish Immunol. 2000 May;10(4):329-41. doi: 10.1006/fsim.1999.0243.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Bass / immunology*
  • Cell Separation / veterinary
  • Digestive System / cytology*
  • Digestive System / immunology
  • Flow Cytometry / veterinary
  • Immunity, Mucosal
  • Immunoglobulin Variable Region / chemistry
  • Immunoglobulin Variable Region / genetics
  • Immunomagnetic Separation / veterinary
  • Lymphoid Tissue / cytology
  • Molecular Sequence Data
  • Phylogeny
  • Receptors, Antigen, T-Cell, alpha-beta / chemistry
  • Receptors, Antigen, T-Cell, alpha-beta / genetics
  • Reverse Transcriptase Polymerase Chain Reaction / veterinary
  • T-Lymphocytes / immunology*

Substances

  • Immunoglobulin Variable Region
  • Receptors, Antigen, T-Cell, alpha-beta