Hagens G, Galley Y, Glaser I, Davis W C, Baldwin C L, Clevers H, Dobbelaere D A
Institute of Animal Pathology, University of Berne, Switzerland.
Gene. 1996 Mar 9;169(2):165-71. doi: 10.1016/0378-1119(95)00769-5.
CD3 epsilon and the zeta-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 CD3 epsilon 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 CD3 epsilon clearly show a higher degree of conservation than the extracellular domains. Bovine CD3 epsilon produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3 epsilon. A partial bovine TCR zeta-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 TCR zeta-chain cDNA sequence. A comparison of the predicted TCR zeta-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCR zeta-chains. In contrast to CD3 epsilon, the short extracellular domain of the TCR zeta-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity. Ab were raised against the TCR zeta-chain, produced as a glutathione S-transferase fusion protein in E. coli, and were used in Western blot analysis to further characterise TCR zeta-chain expression in T-cells. The regents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells.
牛T细胞受体（TCR）的CD3ε和ζ链是两个恒定分子，在通过TCR/CD3复合物进行信号转导中起重要作用。测定了包含完整编码序列的牛CD3ε cDNA克隆的核苷酸序列，并将推导的氨基酸（aa）序列与其他物种的进行了比较。不同CD3ε的胞质结构域比胞外结构域显示出更高程度的保守性。使用不同细菌表达载体在大肠杆菌中产生的牛CD3ε被针对人CD3ε胞质结构域的抗体（Ab）识别。使用基于不同物种间保守序列的引物通过聚合酶链反应（PCR）生成了部分牛TCRζ链cDNA；进行3'和5' RACE-PCR以获得完整的TCRζ链cDNA序列。预测的TCRζ链aa序列比较表明，在其他物种中保守的GDP/GTP结合基序在牛和羊的TCRζ链中显示出明显差异。与CD3ε相反，TCRζ链的短胞外结构域在不同物种间100%保守，跨膜结构域也显示出高度同一性。针对在大肠杆菌中作为谷胱甘肽S-转移酶融合蛋白产生的TCRζ链制备了抗体，并用于蛋白质印迹分析以进一步表征TCRζ链在T细胞中的表达。这些试剂为研究正常和转化的牛T细胞中的信号转导途径提供了有价值的工具。
