Molecular Cloning And Expression Of Fish IL-1Ra-like Gene

Interleukin-1 is a potent mediator of inflammation, involved in regulating a wide variety of physiological and cellular events.The interleukin-1 family arised in vertebrates consists of 11 known genes encoding ligands(IL-1F1 to IL-1F11),whose activities are dependent on heterodimeric receptors of the IL-1R superfamily.phylogenetic analyses indicates that IL1F5 and IL-1F10 are most closely related to IL-1Ra, it also suggested that IL-F5 and IL-1F10 may have diverged from a common ancestor with IL-1ra. Recent research found that IL-1F5 can mediates anti-inflammatory activity through induction of IL-4 following interaction with SIGIRR. And we have already cloned DIGIRR, a SIGIRR orthologue in fish, but to date, no orthologue to IL-1Ra is found in fish or other low vertebrates.In this study, we develop bioinformatic methods based on genomic databases to predict IL-1Ra-like and DIGIRR genes in some fish. Then, the molecular cloning, evolution analysis of TnIL-1Ra-like and southern blotting, eukaryotic expressing of DIGIRR have been done. A recombinant TnIL-1Ra-like protein was also obtained.TnIL-1Ra-like cDNA contains a 591bp open reading frame (ORF). The peptide deduced from ORF is 196 aa in size and weights 22.4 kD. Structurally, TnDIGIRR has a IL-1 domain which conserved in all IL-1 family members. Gene organization of TnIL-1Ra-like is similar to mammalian; they all have 4 coding exons, while the first coding exon of TnIL-1Ra-like is longer. Phylogenetic analysis grouped TnIL-1Ra-like with other predicted fish IL-1Ra-like on a separated branch, excluded from mammalian IL-1Ra. A recombined plasmid TnIL-1Ra-like-pET28a was obtained by sub-cloning the ORF of TnIL-1Ra-like into prokaryotic expression plasmid pET28a and sequenced. Target 26.2Kd protein with his-tag was expressed and purified.All of these lay solid foundation for the subsequent biological function and evolution analysis of genes.