Study On The Structural And Functional Characteristics Of TLR19 In Squaliobarbus Curriculus

Grass carp hemorrhage disease which is caused by Grass carp reovirus(GCRV),has seriously hindered the healthy development of grass carp’s industry.Barbel chub(Squaliobarbus curriculus)and grass carp(Ctenopharymgodon idellus)both belong to the subfamily Leuciscinae,and show stronger resistance to GCRV than grass carp.Toll-like receptor(TLR),as a pattern recognition receptor,can recognize pathogen-associated molecular patterns to activate the immune response.In this study,the TLR19 gene was cloned and identified from barbel chub,and research on its structural and anti-GCRV functional properties were discussed.The main results are as follows:1.cDNA cloning and sequence analysis of ScTLR19The full-length cDNA of ScTLR19 was 3230 bp,including 84 bp 5′-UTR,2871 bp ORF and 275 bp 3′-UTR,encoding 957 amino acids.Expasy predict that the molecular weight of ScTLR19 protein was 109.528 k D and theoretical isoelectric point was 5.99.Blast alignment and phylogenetic tree showed that ScTLR19 shared high similarity and identity with Ci TLR19.ScTLR19 had no signal peptide.SMART domain prediction showed that ScTLR19 comprised Toll/interleukin-1 receptor domain(TIR),transmembrane domain(TM)and nine leucine-rich repeats(LRR).The3 D structure of ScTLR19 owned 15 α-helices and 17 β-sheets.These structural results laid a theoretical foundation for its functional characteristics.2.the expression analysis of ScTLR19ScTLR19 gene was expressed in all 8 healthy tissues(intestine,liver,muscle,skin,spleen,gill,kidney and head kidney).The highest expression was in skin and muscle,followed by head kidney and kidney,and lower expression in intestine,liver,gill and spleen.After GCRV infection,the expression level of ScTLR19 in kidney,spleen and liver showed a trend of first increasing and then decreasing,furthermore they all had a highest expression peak.In addition,compared with the control group,the expression level of ScTLR19 in kidney was significantly up-regulated,and there was no significant difference in spleen,while in liver was significantly down-regulated at 12 h,72 h and 168 h in experimental groups.In conclusion,ScTLR19 might mainly play an immune function in kidney as a PRR gene after GCRV stimulation.3.the characteristic analysis of ScTLR19 anti-GCRV immune function(1)When Squaliobarbus curriculus fin cells overexpressed wild-type ScTLR19 and were then challenged with GCRV,the transcriptional expression of interferon regulatory factor 7(IRF7)did not significantly change,while the transcriptional expression of interferon regulatory factor 3(IRF3)and both My D88 and TRIF that were reported as two important adaptor proteins in TLR signal pathways,were significantly up-regulated at 12 h and 24 h post GCRV infection,compared to the control group(P<0.05).ScTLR19 only regulated IRF3 but not IRF7 signal pathway during GCRV infection.In addition,ScTLR19 could regulate both My D88 and TRIF signal pathways,which might enable ScTLR19 with more antiviral immune functions to enhance the resistance against GCRV for barbel chub.(2)When Squaliobarbus curriculus fin cells overexpressed deletion ScTLR19 and were then challenged with GCRV,the transcriptional expression of IRF3、IRF7、TRIF and My D88 tended to be down-regulated and then increased from 12 h to 48 h;while Squaliobarbus curriculus fin cells overexpressed wild-type ScTLR19,the transcriptional expression of IRF3、TRIF and My D88 were initially up-regulated and subsequently decreased from 12 h to 48 h.The structural characteristics of extracellular LRR might determine its immune recognition function.These results provided clues for in-depth analysis of the different strategies and mechanisms under the stimulation of GCRV between barbel chub and grass carp.