Functional Characterization Of T-cell Activation-related Genes In Nile Tilapia, Oreochromis Niloticus

Tilapia(Oreochromis spp.) is one of the most important economical fish and widely cultured throughout the world. In recent years, infectious diseases of tilapia, espcially the streptococcosis, have been severe, resulting in great economic loss and threatening the development of tilapia aquaculture. Because of its high commercial interest, extensive researches on the diseases which caused high mortality were carried out, and S. agalactiae was confirmed as its main causative agent. However, fewer studies focused on T-cell of tilapia, especially in the area of T-cell activation. CD59, CD2BP2 and Lck, three kinds of key molecules interacting with costimulatory molecule CD2, play a crucial role in CD2-triggered T-cell activation. In this study, to better understand the mechanism of T-cell response to intracellular infection of bacteria in tilapia, the full length of CD59, CD2BP2 and Lck c DNA were cloned from Nile tilapia, Oreochromis niloticus, and its tissue distribution, expression profile in response to S. agalactiae stimulus and functional properties were investigated.CD59, functioning as a complement inhibitor and T-cell regulator, plays an important role in both innate and adaptive immune responses. In this paper, the CD59 gene of Nile tilapia, O. niloticus(designated as On-CD59) was cloned. Sequence analysis showed that the full length of On-CD59 c DNA was 1176 bp, containing a 5’-untranslated region(UTR) of 74 bp, a 3’-UTR of 748 bp and an open reading frame(ORF) of 354 bp which is encoding 117 amino acids. Five important structural features, including a signal peptide at the N-terminus, a LU domain with the consensus motif CCXXXXCN, a GPI-anchoring region at the C-terminus, and 10 cysteine residues were detected in the deduced amino acid sequence of On-CD59. In healthy tilapia, the On-CD59 transcripts could be detected in all the examined tissues, with the most abundant expression in the brain. While vaccinated with inactivated S. agalactiae 24 h later, the On-CD59 m RNA expression was significantly up-regulated in the skin, brain, head kidney, thymus and spleen. Moreover, there was a clear time-dependent expression pattern of On-CD59 in the skin, brain, head kidney, thymus and spleen after immunization and the expression reached the highest level at time points of 12 h in the skin, 24 h in the brain and head kidney, and 48 h in the thymus and spleen, respectively. The assays for the complement-inhibitory activity suggested that recombinant On-CD59 protein had a species-selective inhibition of complement. In addition, by biotin-streptavidin ELISA and colony-forming unit assay, we demonstrated that recombinant On-CD59 protein may possess both binding activities to PGN and LTA, which are two main signature molecules in cell wall of Gram-positive bacteria, and inhibiting activity of S. agalactiae.CD2BP2, one of several proteins interacting with the cytoplasmic tail of CD2, plays a crucial role in CD2-triggered T cell activation and nuclear splicing. In this paper, the CD2BP2 gene of Nile tilapia, O. niloticus(designated as On-CD2BP2) was cloned. Sequence analysis showed that the full length of On-CD2BP2 c DNA was 1429 bp, containing a 5’-UTR of 111 bp, a 3’-UTR of 193 bp and an ORF of 1125 bp which is encoding 374 amino acids. Two important structural features, a GYF domain and a consensus motif GPFXXXXMXXWXXXGYF were detected in the deduced amino acid sequence of On-CD2BP2, and the deduced genomic structure of On-CD2BP2 was similar to the known CD2BP2. In healthy tilapia, the On-CD2BP2 transcripts were mainly detected in the head kidney and spleen. While vaccinated with inactivated S. agalactiae 48 h later, the On-CD2BP2 m RNA expression was significantly up-regulated in the head kidney, spleen and brain. Moreover, there was a clear time-dependent expression pattern of On-CD2BP2 in the head kidney, spleen and brain after immunization and reached the highest level at time points of 24 h in the brain, and 48 h in the head kidney and spleen, respectively.Lck plays a critical role in effective signal transductions that are fundamental to T-cell differentiation, proliferation and effector functions. In this paper, the Lck gene of Nile tilapia, O. niloticus(designated as On-Lck) was cloned. Sequence analysis showed Sequence analysis showed that the full length of On-CD59 c DNA was 2213 bp, containing a 5’- UTR of 286 bp, a 3’-UTR of 421 bp and an ORF of 1506 bp which is encoding 501 amino acids. Several important structural characteristics required for TCRs signal transduction were detected in the deduced amino acid sequence of On-Lck, and the deduced genomic structure of On-Lck was similar to the known Lck. In healthy tilapia, the On-Lck transcripts were mainly detected in the thymus, spleen, head kidney and gill. When immunized with inactivated S. agalactiae, the On-Lck m RNA expression was significantly up-regulated in the thymus, spleen and head kidney. Moreover, there was a clear time-dependent expression pattern of On-Lck in the thymus, spleen and head kidney after immunization and the expression reached the highest level at 48 h in the spleen and thymus, and at 72 h in the head kidney, respectively.