Evolution And Expression Analysis Of Tumor Necrosis Factor 15 (TNFSF15/TL1A) And Its Receptor 25 (TNFRSF25) In Grass Carp (Ctenopharyngodon Idella)

The tumor necrosis factor superfamily(TNFSF)of ligands and receptors(TNFRSF)are fundamental for many biological processes such as immunity,inflammation and hematopoiesis.To date,19 TNFSFs and 29 TNFRSFs were discovered in humans;14TNFSFs were identified in teleost fish.The TNFSF15/TL1A was first reported in 2002by Migone et al.and contained four exons and three introns.The TNFRSF25(also known as DR3,Wsl-1,TR3,Apo-3,TRAMP,TNFRSF12)was first reported in 1996when Kitson et al.exploited sequences in the human genome.The sequences are homologous to TNFR1 and contain a death domain.Grass carp(Ctenopharyngodon idella)is the most important economic fish with a long history in China's aquaculture industry.Grass carp disease,however,has always hindered the development of our countery's aquaculture industry.The research on the immune inflammation of grass carp is bouth important for actual production and development of immune theory.In this study,the CiTNFSF15/TL1A and CiTNFRSF25 genes were cloned and identified.The expression level of CiTNFSF15/TL1A and CiTNFRSF25 in different tissues could be detected by the infection experiment of F.cloumnare and GCRV.The recombinant proteins of the target genes were expressed and purified using E.coli prokaryotic expression technology.The following results were mainly obtained:1.TNFSF15/TL1A(termed CiTNFSF15/TL1A)and TNFRSF25(termed CiTNFSF25)genes of grass carps were cloned and identified,respectively.The full-length cDNA of CiTNFSF15/TL1A was 1095 bp,including a 723 bp open reading frame and encoding a peptide of 240 aa.The protein sequence of CiTNFSF15/TL1A contains a TM region(36-58 aa)and a THD region(98-237 aa).The full-length cDNA of CiTNFRSF25 is 1420 bp,including a1152 bp open reading frame and encoding a peptide of 383 aa.Ci TNFRSF25 protein sequence contains an N-terminal signal peptide(1-22 aa),a TM region(173-195 aa)and a DD region(291-371 aa).Further analysis shows that the CiTNFRSF25 is an orthologue of fish TNFRSF1ALs and shares conserved gene synteny with human TNFRSF25.2.Real-time fluorescence quantitative(RT-qpcr)results showed that TNFSF15/TL1A and TNFRSF25 genes were constitutively expressed in the brain,skin,gill,intestine,thymus,trunk kidney,liver,head kidney and spleen of grass carp.The expression of CiTNFSF15/TL1A and CiTNFRSF25 can be modulated by infection of F.cloumnare and GCRV.The CiTNFSF15/TL1A and CiTNFRSF25 were significantly down-regulated(P<0.01)in both kidney and spleen at 24 hpi.with 1×10~7 cfu/mL F.cloumnare.3.The recombinant proteins of CiTNFSF15/TL1A(rCiTNFSF15/TL1A)(24KDa)and CiTNFRSF25(rCiTNFRSF15)(19 KDa)were expressed and purified,respectivetly.The results of protein activity assay revealed that when the rCiTNFSF15/TL1A protein co-incubated with primary kidney cells for 24 h,it would regulate the secretion of CiTNFRSF25,c-IAP,IL-1?,IFN-?,Caspase-7 and TNF-?.