Cloning, sequencing and expression of the bovine CD3ε and TCR-ζ chains, two invariant components of the T-cell receptor complex

CD3ε and the ζ-chain of the bovine T-cell receptor (TCR) are two invariant molecules with an important role in signal transduction via the TCR/CD3 complex. The nucleotide sequence of a bovine CD3ε cDNA clone containing the complete coding sequence was determined and the deduced amino acid (aa) sequence compared to that of other species. The cytoplasmic domains of the different CD3ε clearly show a higher degree of conservation than the extracellular domains. Bovine CD3ε produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3ε. A partial bovine TCRζ-chain cDNA was generated by the polymerase chain reaction (PCR) using primers that were based on sequences that are conserved between different species; 3' and 5' RACE-PCR were carried out to obtain the complete TCRζ-chain cDNA sequence. A comparison of the predicted TCRζ-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCRζ-chains. In contrast to CD3ε, the short extracellular domain of the TCRζ-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity. Ab were raised against the TCRζ-chain, produced as a glutathione S-transferase fusion protein in E. coli, and were used in Western blot analysis to further characterise TCRζ-chain expression in T-cells. These reagents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells.