Identification and functional characterization of interleukin-12 receptor beta 1 and 2 in grass carp (Ctenopharyngodon idella).

Interleukin 12 (IL-12) binds its receptor complex of IL-12 receptor beta 1 (IL-12Rß1) and IL-12Rß2 to transduce cellular signaling in mammals. In teleosts, the function of Il-12 is drawing increasing attention, but molecular and functional features of Il-12 receptors remain obscure. Especially, the existence of multiple Il-12 isoforms in some fish species elicits the requirement to clarify their receptors. In this study, we isolated three cDNA sequences as Il-12 receptor candidates from grass carp, entitled as grass carp Il-12rß1 (gcIl-12rß1), gcIl-12rß2a and gcIl-12rß2b. In silico analysis showed that gcIl-12rß1 and gcIl-12rß2a shared the conserved gene locus and similar structure characteristics with their orthologues of zebrafish, frog, chicken, mouse and human, respectively. However, the Il-12rß2b of grass carp and zebrafish was similar to IL-27Ra in non-fish species. Further locally installed BLAST and gene synteny analysis uncovered three gcIl-12 receptors being single copied genes. Tissue distribution assay revealed that gcil12rß1 and gcil12rß2a transcripts were predominantly expressed in head kidney, differing from the even distribution of gcil12rß2b transcripts in all detected tissues. Subsequently, the binding ability and antagonistic effects of recombinant extracellular region of gcIl-12rß1 with recombinant grass carp Il-12 (rgcIl-12) isoforms were explored, providing functional evidence of the newly cloned gcIl-12rß1 being genuine orthologues of mammalian IL-12Rß1. Moreover, our data showed that gcIl-12rß1 and gcIl-12rß2a but not gcIl-12rß1 and gcIl-12rß2b mediated the effects of rgcIl-12 isoforms on ifn-γ promoter activity, thereby revealing Il-12 receptor signaling in fish. These results identified grass carp Il-12 receptors, thereby advancing our understanding of Il-12 isoform signaling in fish.

Molecular characterization, tissue distribution and expression analysis of interleukin-12 receptor ß2 chain in sheep.

Ovine ß2 subunit of the interleukin (IL)-12 receptor (IL-12Rß2) was cloned from mRNA preparation of mitogen-activated peripheral blood mononuclear cells (PBMCs). The complete coding sequence for ovine IL-12 Rß2 was found to be 2586 nucleotides in length encoding 862-amino-acid residue protein. It showed 96.4% homology at the nucleotide level and 94.1% homology at the amino acid level with bovine IL-12 Rß2. The ovine IL-12 Rß2 subunit shares common structural and functional elements with their counterparts from the other species. Phylogenetic tree showed that ovine IL-12Rß2 was clustered into the Artiodactyla group, together with those of cattle and pig, which was distinct from the other groups. Real-time RT-PCR was used to investigate expression of the IL-12Rß2 in different tissues of sheep in order to determine the characterization of this receptor in tissue. Expression analysis showed that IL-12Rß2 mRNA expression was detected at all the detected tissues with the exception of thymus.