Molecular cloning and characterization of common carp ( Cyprinus carpio L.) TCRγ and CD3γ/δ chains

Abstract

Partial cDNA sequences of TCRγ and CD3γ/δ were isolated from the thymus of common carp ( Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCRγ and CD3γ/δ were obtained by means of 3′ RACE and 5′ RACE, respectively. The full length of carp TCRγ chain is 1368 bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCRγ contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCRγ. The C region of carp TCRγ contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR Cγ contains 37 amino acids. The full length of carp CD3γ/δ is 790 bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3γ/δs, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3γ/δ. Differing from other known CD3γ/δs, carp CD3γ/δ lacks the CXXCXE motif in the extracellular domain. RT-PCR analysis demonstrated that the expression of TCRγ gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCRγ gene was detected in all the examined tissues. The expression of CD3γ/δ gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3γ/δ-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone.