Molecular Clonign And Expression Analysis Of DIGIRR From Golden Pompano (Trachinotus Ovatus)

SIGIRR(Single Ig IL-1R related molecule)is an important member of the Toll/IL-1R superfamily,which can compete with TLRs to bind to adaptor molecules and inhibit the excessive inflammatory response via TLRs signaling pathway,playing a crucial role in regulating mammalian innate immunity and autogenic diseases.DIGIRR(Double Ig IL-1R related molecule)is a mammalian SIGIRR homologue,only presents in fish,which can negatively regulate the inflammatory response induced by IL-1 or LPS,and play a key role in regulating fish immune response.Golden pompano(Trachinotus ovatus),as a marine fish specie with economical value,is widely cultured in the Southeast coastal areas of China.With the expanding of intensive aquaculture and pollution of the aquatic environment,a variety of diseases caused by bacteria,viruses and parasites have broken out in golden pompano farming and resulted in severe economical losing.Understanding the molecular characteristics,immune response mechanism and protein functions of DIGIRR in golden pompano will contribute to new strategies for disease prevention and breeding of disease resistance.In this study,the DIGIRR(TroDIGIRR)gene from golden pompano was identified.Furthermore,the TroDIGIRR sequence structure,normal tissues expression,immune responses,subcellular localization,exploring interaction molecules and polyclonal antibodies preparation were investigated.The main results obtained are as follows:1.The full length cDNA of TroDIGIRR was 2167 bp with a 1572 bp of ORF encoding 523 amino acids.Protein structure analysis showed that TroDIGIRR contained a signal peptide,two Ig domains,a transmembrane region and a conserved TIR domain.TroDIGIRR shared a higher protein sequence identity with other ichthyic DIGIRR(68.5%-86.3%)and a moderate identity with fish SIGIRR(54.1%-68.5%).Meanwhile,the Ig domains and the TIR domain of TroDIGIRR exhibited 57.3%-87.6%identity and 76.2%-92.5% identity to the corresponding domains from other fish DIGIRR,respectively.Furthermore,TroDIGIRR was properly clustered with other teleost DIGIRR on the NJ phylogenetic tree.These results suggested that TroDIGIRR was exactly the homologue of vertebrates’ DIGIRR,which was highly conserved in the evolutionary process.2.qPCR analysis found that TroDIGIRR was constitutively expressed in all the examined normal tissues(brain,gill,skin,muscle,intestine,heart,liver,spleen and head kidney)from healthy fish,most highly in the intestine,followed by the liver,moderately in the head kidney and spleen,and lowly in the heart,which suggested that TroDIGIRR was involved in physiological activities.3.The expression changes of TroDIGIRR were investigated in the immune-related tissues(head kidney,spleen,gill,intestine and liver)following LPS,poly I:C and Vibrio alginolyticus stimulation.The results were as follows:(1)After LPS challenge,the expressions levels of TroDIGIRR in the head kidney,liver,intestine and gill were significantly down-regulated at 6 h,but prominently increased at 12 and 24 h contrasted with the control group(P<0.05).After stimulation 48 h,the expression in the liver,intestine and gill restored normal levels,while the expression in the head kidney was still up-regulated.The expression levels of TroDIGIRR in the spleen was peaked at 12 h and then gradually decreased to normal levels.(2)Following poly I:C challenge,the expression levels of TroDIGIRR was markedly down-regulated in the intestine at 6 h,but continuously increased between 12 h to 48 h.The expression levels of TroDIGIRR in the liver was strikingly down-regulated at 6 h and peaked at 12 h after stimulation,then began to decrease,but still dramatically enhanced compared with control group(P<0.05).The expression of TroDIGIRR in the spleen was significantly induced at 12-48 h;which in the head kidney was consistent with that of spleen.The expression of TroDIGIRR in gill was no significant changes at all time points except for induced at 24 h.(3)After Vibrio alginolyticus infection,the expression levels of TroDIGIRR in the head kidney and intestine were markedly down-regulated at 6 h post infection,but significantly up-regulated at 12 h and 24 h,respectively.The expression levels of TroDIGIRR in the spleen was significantly down-regulated at 6h,while strikingly up-regulated at other time points.The expressions of TroDIGIRR in the liver and gill were prominently increased at 12 h,with no significant changes at other time points.These results suggested that TroDIGIRR not only play crucial immune role during bacterial infection,but also may participate in antiviral immune response.4.The recombinant TroDIGIRR extracellular and intracellular domains(pET32a-DIG-ECDs and pET32a-DIG-TIR)proteins were obtained by using pET-32 a prokaryotic expression system and refolded by dialysis,and then immunized Japanese white rabbits and collected serums.Western blot showed the antiserum were reacted specifically with pET32a-DIG-ECDs and pET32a-DIG-TIR proteins,respectively.ELISA analysis showed the titers of anti-DIG-ECDs and anti-DIG-TIR serums were 1:256k and 1:2048k,respectively.The antibodies against DIG-ECDs and DIG-TIR proteins will facilitate to screen the TroDIGIRR possible ligands and biological function.5.The eukaryotic vector pEGFP-N1-TroDIGIRR was constructed and transfected into HEK-293 T cells.After 48 h,the cell were stained with DAPI and TroDIGIRR protein mainly distributed in the cytoplasm was observed by fluorescence microscopy.6.The Eukaryotic recombinant plasmids were constructed and co-transfected into HEK-293 T cells.After 48 h,the total protein was extracted from cells and investigated protein-protein interaction by using co-immunoprecipitation and western blot assay,which showed TroDIGIRR could interact with TroMy D88,but not with TroTRIF.