Cloning And Expression Of Several Marker Genes Of T Lymphocytes In Tilapia

T lymphocytes can mediate the specific cellular immune response in the body, and play an important role in the immune response and regulation. Teleost fish are the most primitive vertebrates with both non-specific and specific immune response. The research about teleost immune not only contributes to the understanding of the immune molecules from the perspective of molecular evolution, but also has an important position and role in fish disease control. On one hand, a large number of immune cell associated molecules in teleost have been successively isolated and identified, which substantially promotes the molecular immunology research of teleost. On the other hand, due to the deficiency of specific antibodies to lymphocyte molecular makers, the diversity and species specific of teleost, therefore, the study of the subsets, differentiation and development, function and regulation mechanisms of T lymphocytes are still in infancy. Till to now, T lymphocytes molecular makers of tilapia which is the worldwide farmed fish, have not been cloned and identified. In view of this, on one hand, T lymphoctye-specific differentiation antigens CD3y/8, CD4, CD8a cDNA sequence from tilapia (in this study, tilapia was assigned as Nile tilapia (Oreochromis niloticus)) if there has no special explanation) were cloned and identified; on the one hand, the prokaryotic expression vectors of part section of CD3y/8, CD4, CD8a and Foxp3 from tilapia were constructed, and then their prokaryotic expression conditions were investigated, respectively. Following results were achieved:1. Combination of bioinformatics analysis and RT-PCR, tilapia CD3γ/δ, CD4 and CD8a cDNA sequences were successfully cloned and analyzed. The details were as follows:1) The cDNA of tilapia CD3y/8, constituted by six exons, was 620 bp, which included 86 bp 5’UTR,18 bp 3’UTR and 516 bp ORF coding a 171-amino acid polypedtide. The pupative primary structure of the CD3γ/δpolypedtide contained a signal peptide of 27 amino acids (aa) followed by a extracellular domain of 68 aa, a transmenbrane of 53 aa and a cytoplasmic tail of 23 aa. To explore the relationships between tilapia CD3γ/δand other CD3 molecules including CD3y, CD38 and CD3ε, phylogenetic tree was construsted based on the coding sequeneces of CD3. In the phylogenetic tree, CD3y/5, CD3y, CD38 and CD3s were clustered as distinct branches by ClustalW program, while the branch between CD3εand other three groups was relatively long, suggesting that CD3y/8, CD3y, CD38 may not share the ancestral gene with CD3s. Meanwhile, tilapia CD3y/8 was clustered with other species CD3y/8, such as Mandarin fish(Siniperca chuats), Atlantic salmon(Salmo sala), Flounder(Paralichthys olivaceus), Fugu(Takifugu rubripe) and Tetraodon(Tetraodon nigroviridis) as one clade, which showed amino acid sequence similarity of 56.4%,44.4%,43.6%,52.5%,51.2%, respectively. The primary structure analysis showed that tilapia CD3y/8 shared characteristics with other subtypes of CD3, which was the two cysteine residues and CxxCxE motif of the extracellular region, and a cytoplasmic immune receptor activation motif IT AM.2) The cDNA of tilapia CD4, constituted by nine exons, was 3259 bp, which included 141 bp 5’UTR,1714 bp 3’UTR and 1404 bp ORF coding a 467-amino acid polypedtide. The pupative primary structure of the CD4 polypedtide contained a signal peptide of 20 aa followed by a extracellular domain of 394 aa, a transmenbrane of 23 aa and a cytoplasmic tail of 30 aa. Phylogenetic analysis showed that CD4 and CD4-like were clustered as different branches, suggesting that CD4 may not share the ancestral gene with CD4-like. The tilapia CD4, was clustered with other species CD4, such as Fugu, Tetraodon, Rainbow trout(Oncorhynchus mykiss), Channel Catfish (Ictalurus punctatus) and Zebrafish(Danio rerio) as one clade, which showed amino acid sequence similarity of 45.8%,46%,37.6%, 34.5% and 29.3%, respectively. The structure analysis indicated that tilapia CD4 contained four immunoglobulin-like domains in the extracellular region, which included the WTC motif and the N-glycosylation sites, and the cytoplasmic domain with a conserved CxC motif. 3) The cDNA of tilapia CD8a, constituted by five exons, was 1050 bp, which included 424 bp 5’UTR,626 bp coding sequences encoding a 208-amino acid polypedtide. The pupative primary structure of the CD8 polypedtide contained a signal peptide of 20 aa followed by a extracellular domain of 394 aa, a transmenbrane of 23 aa and a cytoplasmic tail of 30 aa. The pupative primary structure of the CD8a polypedtide contained a signal peptide of 21 aa followed by a extracellular domain of 151 aa, a transmenbrane of 23 aa and a cytoplasmic tail of 13 aa. Phylogenetic analysis indicated that CD8a and CD8P were clustered as different clades, suggesting that CD8a may not share the ancestral gene with CD8p. Meanwhile, tilapia CD8a was clustered with other species CD8a, such as Fugu, Atlantic salmon, Channel Catfish, Zebrafish, Carps(Cyprinus carpio) as one clade, which showed amino acid sequence similarity of 51.7%,41.8%,34.6%,32.3%, 35.9%, respectively. Further structural analysis indicated that the two cysteine motif involved in disulfide bond formation in the V domain was very conservative, while the p56lck binding motif CxC and residues interacting withβ2m and MHC I such as arginine, lysine and leucine were not conservative.2. RT-PCR was used to detect the transcripts of tilapia CD3γ/δ, CD4 and CD8a in different tissues. The results showed that CD3γ/δ, CD4 and CD8a mRNA could be detected in most tissues, while the higher level in immune related tissues such as gill, spleen, kidney, head kidney, intestine was detected.3. Combination bioinformatics analysis and the fusion protein prokaryotic expression strategy, the cDNA sequences encoding extracellular domains of tilapia CD3γ/δ, CD4, CD8a and Foxp3 defiency of the conserved forkhead domain, whose homologue in mammals was known as the specific markers of regulatory T lymphocytes, were inserted to prokaryotic expression vector pCold I and pET-28a, respectively. Expression of target genes was induced by IPTG in different conditions and confirmed by SDS-PAGE, the results were as follows:1) The recombinant plasmids CD3γ/δ-pET-28 could be induced to express fusion protein which existed in supernatant at 20℃,30℃and 37℃, the highest level of expression occurred at 37℃.2) The recombinant plasmid Foxp3-F1R2-pET-28 could be induced to express fusion protein which existed in inclusion body at 30℃and 37℃, and the level of expression had no significant difference. 3) The recombinant plasmid CD4-pCold I, CD8-pET-28 CD4-pET-28a were not expressible under different conditons.