Molecular Cloning And Identification Of IL-1β And IL-1R1-like In Teleost Fish

Interleukin-1 plays an important role in the local and systemic inflammatory response;including IL-1αand IL-1β(named as IL-1F1 and IL-1F2,respectively).At present,the Interleukin-1 family arised in vertebrates consists of 11 known genes encoding ligands(IL-1F1 to IL-1F11).But to date,no orthologue to IL-1βis found in fish or other low vertebrates.IL-1 functions by binding their receptors,IL-1 Receptor,to mediate signal transduction to realize their biological effects.IL-1 Receptor family contains typeⅠreceptors(IL-1R1),typeⅡreceptors(IL-1R2),and the IL-1 receptor accessory protein(IL-1RAP).In this study,we developed bioinformatics methods based on genomic databases, successfully cloned IL-1βgene and IL-1R-like gene in Tetraodon,and obtained recombinant TNIL-1βprotein and rabbit anti-IL-1βserum.TNIL-1βcDNA contains 4 exons,3 introns and a 774bp open reading frame(ORF).The peptide deduced from ORF is 257aa in size.TNIL-1βpromoter comprises several transcription binding sites, such as TATA-box,NF-KB,CEBP,AP1,CCAAT,and so on,3'UTR contains 5 ATTTA instability motif.A recombined plasmid TNIL-1β-pET41a was obtained by sub-cloning the ORF of TNIL-1βinto prokaryotic expression plasmid pET41a and sequenced.Target 60.1Kd protein was expressed after IPTG induce and purified by Ni-NTA.TNIL-1R-like gene contains 11 exons,and a 1902bp ORF,coding 633aa, which has 3 Ig Domain,1 transmembrane domain(TMD),and 1 TIR Domain.All the results lay foundation in function studies of fish IL-1βand IL-1R,and is important for fish immune system research and evolution of immunology genes.