Molecular Cloning, Expression Analysis Of ILF2 And IL-21 Homologue From Tetraodon Nigroviridi

By utilizing the Tetraodon nigroviridi genome database and kinds of biologic softwares, we successfully cloned two novel immune genes, Tetraodon nigroviridi ILF2 homologue (TnILF2) and Tetraodon nigroviridi IL-21 homologue (TnIL-21 ) from Tetraodon nigroviridi.The full-length TnILF2 cDNA is 1380 bp in size and comprised of a 5'UTR of 57 bp, a coding region of 1164 bp and a 3'UTR of 159 bp. The TnILF2 peptide deduced from ORF is 387 amino acids in size and weights 42.9 kD. The TnILF2 peptide contains 2 N-glycosylation sites, several phosphorylation sites, 1 RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain. Multiple sequences analysis shows that the TnILF2 peptide share 58% ⁹3 % amino acid identities with other known ILF2 moleculars. Constitutive expression of Tetraodon ILF2 was observed in all of the 7 tissues examined.stimulation with LPS did not significantly alter the expression of Tetraodon ILF2. A recombined plasmid TnILF2-pcDNA3.1+ was obtained by sub-cloning the ORF of TnILF2 into eucokaryotic expression plasmid pcDNA3.1 + and sequenced.The full-length TnIL-21 cDNA is 849 bp in size and comprised of a 5'UTR of 69 bp, a coding region of 438 bp and a 3'UTR of 342 bp. The TnIL-21 peptide deduced from ORF is 145 aa in size and weights 16.5 kD. contains several modify sites and function domains were found within TnIL-21 peptide, including three N-glycosylation sites, several phosphorylation sites, a leucine-zipper, a N-terminal signal composed of 22 amino acids. The mature TnIL-21 also contains four α-helixes, the IL-2 family signature motif, four cysteine residues (Cys⁶¹, Cys⁶⁸, Cys¹¹⁰ and Cys¹¹³) and a glutamine (Gln¹³³). Multiple sequences analysis shows that the TnIL-21 peptide shares 20%⁴9% amino acid identities with other known IL-21 molecules. Low constitutive expression of TnIL-21 was observed in limited tissues of healthy fish including gut, gill and gonad. Stimulation with LPS significantly induced the expression of TnIL-21 in kidney and spleen. Compared genes up- and down-stream IL-21 between Tetraodon nigroviridi, Fugu rubripes and human, a high degree of conservation of synteny was found. A recombined plasmid TnIL21-pET32b was obtained by sub-cloning the ORF of TnIL-21 into prokaryotic expression plasmid pET32b and sequenced. Sequencing result showed that both of the sequence and reading frame were correct. Target protein was expressed by induction using IPTG.In summary, two novel genes were successfully cloned and the expression plasmids were recombined. This work will pave the way for the further investigation on the biological function.