| As co-receptor and signal molecular expressed on surface of T cells and thymocytes in mammalian species,CD3,CD4 and CD8 molecules are involved in the activation of peripheral T cells and in the differentiation of thymocytes.In this study, the full-length cDNAs of porcine CD3e,CD4 and CD8a were amplified and cloned from total RNA of the lymphocytes and peripheral blood lymphocytes by RT-PCR and RACE(rapid amplification of cDNA ends, RACE), and then the characteristics of cDNAs sequences and predicted amino acid sequences of porcine CD3e, CD4 and CD8a were analyzed by bioinformatics. Additionlly.prokaryotic expression system with fusion protein tag were adopted to perform expression of extracellular domain of porcine CD3e, CD4 and CD8a in Escherichia collSequence analysis revealed that the amplified porcine CD3e cDNA was 1241nt in length, including 80nt of 5'untranslated region, 591nt of open reading frame and 570nt of 3'untranslated region, the open reading frame encoded porcine CD3e protein precursor with 196 amino acids residues lacking N-glycosylation site. Four cysteine residues (Cys49, Cys91, Cys108, Cys111) were conserved among mammalian species within the extracellular domain.The porcine CD3e contained several characteristic motifs highly conserved across the species within the cytoplasmic domain, namely Src homology 3(SH3), endoplasmic reticulum (ER) retention motif and the immunoreceptor tyrosine-based activation motif (ITAM) which consisted of two Src homology 2 (SH2) and two potential tyrosine phosphorylation sites at Tyr177and Tyr188 .These motifs were associated with signal transduction on T-cell activation as well as with assembly and expression of TCR-CD3 complex. Comparison of the deduced amino acids sequence of porcine CD3e with those of human, mouse, dog, cow and sheep showed that the amino acids homology were 61.2%, 58.7%, 58.7%, 65.1% and 65.6%, respectively.It was discovered that there were alternatively spliced transcripts of CD4 mRNA existed in the porcine thymocytes and peripheral blood lymphocytes in this study, and one of the transcripts had a deletion of 120nt coding 40 amino acid residues, suggesting that it could encode a porcine secreted CD4 protein. Amplified porcine CD4 full-length cDNA was 2715nt in length, including 158nt of 5'untranslated region, 1374nt of coding region and 1183nt of 3'un-translated region. The open reading frame encoded porcine CD4 protein precursor with 457 amino acid residues and four N- glycosylation site. Seven cysteine residue (Cys43, Cys122, Cys327, Cys418, Cys421,Cys444 and Cys446) and two serine residues (Ser432 and Ser439) were cqnserved among mammalian species, highly conserved protein tyrosine kinase p56lck recognition site KKTCQC and a di-leucine motif involved in antigen-induced CD4 internalization were in the cytoplasmic domain. Comparison of the deduced amino acids sequence of porcine CD4 with those of human , rabbit, cat, dog and mouse revealeded that the amino acidshomology were 56.0%, 54.5%, 56.9%, 56.5% and 44.9%, respectively.Sequence analysis indicated that the amplified porcine CD8a cDNA was 2179nt in length, including 165nt of 5'un-translated region, 71 lnt of open reading frame and 1303nt of 3'un-translated region.The open reading frame encoded porcin CD8a protein precursor with 236 amino acids residues and one N-glycosylation site. Seven cysteine residues (Cys46, Cys57, Cys118, Cys167,Cys182, Cys21 and Cys218) were conserved among mammalian species, and an highly conserved protein tyrosine kinase p56lck recognition site CPxP was in the cytoplasmic domain. Comparison of the deduced amino acids sequence of porcine CD8a with those of human, cow, mouse, dog, and cat showed that the amino acids homology were 55.7%, 57.6%, 35.6%, 56.8% and 56.4 %, respectively.The genes for extracellular domain of porcine CD3e,CD4 and CD8a were amplified by PCR from recombinant plasmids pGEM-TCD3A, pGEM-TCD4BlL and pGEM-TCD8B, and subcloned into pGEX-4T-2, pGEX-6P-l and pGEX-6P-l , respectively. E. coli BL21 cells were transformed with...
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