The Different Structure And Expression Feature Of TLR2 In Squaliobarbus Curriculus And Ctenopharymgodon Idellus After GCRV Infection

Grass carp hemorrhage disease,which is caused by Grass carp reovirus(GCRV),has seriously hindered development and prosperity of grass carp aquaculture in China.Grass carp Ctenopharymgodon idellus and barbel chub Squaliobarbus curriculus both belong to the subfamily Leueiscinae.Based on the significant difference on the phenotype between grass carp and barbel chub after GCRV infection and the fact that TLRs pathway plays an important role against pathogen invasion,we studied the function of TLRs in the immune response of barbel chub(ScTLR22)and grass carp(CiTLR22)against GCRV.Three genes,which are in TLRs pathway of barbel chub and are closely related to viral infection,were cloned.We also analyzed the expression feature of those three genes after GCRV infection.The methods of molecular biology,cell biology and bioinformatics were used to compare the difference structural and functional of TLR22 gene between grass carp and barbel chub.Results of the study could give an evidence in TLRs pathway about the great different on phenotype between grass carp and barbel chub after GCRV infection.The main results are following: 1 Gene clone and antiviral analysis of three genes in TLRs pathway of barbel chubThe cDNA of TLR22,Myeloid differentiation primary response protein 88(MyD88)and IFN regulation factor 3(IRF3)in TLRs pathway from barbel chub were cloned and the antiGCRV virus effect were studied.Results shown TLR22,MyD88 and IRF3 seem to play a vital role in the immune response against GCRV.In addition,the overexpression of TLR22 and IRF3 in CIK cells could inhibit the replication of GCRV.2 The structural characteristics of ScTLR22 and CiTLR22The results of domain features predicted by the SMART compared with selected Cyprinids shown the last LRR(LRR18)domain of ScTLR22 with 12 amino acids before the LRRCT domain was the same as that of TLR22 s of Danio rerio and Cyprinus carpio,while it was different from that in grass carp,in which the last LRR(LRR17)has 60 amino acids before the LRRCT domain.Multiple amino acid sequences alignments of TIR domain with the Cyprinid TLR22 shown that the amino acid sequences of the TIR domain from Cyprinidae TLR22 exhibited greater sequence conservation and the three particularly conserved regions were observed,which were completely consistent with the tested Cyprinidae fish except grass carp.One amino acid mutation was found in Box 1 and 2,respectively.Are the structural differences in recognition region of ScTLR22 and CiTLR22 lead to differences in function? The model of ScTLR22 and CiTLR22 interaction with poly(I:C)were evaluated by AutoDock 4.0 and Plusplot+ shown: the binding energy,intermolecular energy,Hydrogen bonds and Length of hydrogen bonds of the complexes formed by TLR22 and poly(I:C)were better than that of CiTLR22.Therefore,the combination of ScTLR22 recognition region and poly(I:C)was more close and stable than that of CiTLR22.3 Different abilities of recognizing GCRV and inducing innate immune response of ScTLR22 and Ci TLR22To determine the different functions of ScTLR22 and CiTLR22,pEGFP-ScTLR22,pEGFP-CiTLR22 and pEGFP-ScE/CiC were transfected into CIK cells.After screening of the stable overexpressed cells,the transcript level of TLR22,IRF3,IFN-?,IL-1? and the GCRV titer were evaluated by qRT-PCR and TCID50 method,respectively.After GCRV infection,the IRF3,IFN-? and IL-1? expression levels in CIK cells overexpressing ScTLR22,CiTLR22 and fused cDNA encoding the extracellular domain of ScTLR22(ScE)to the transmembrane and cytoplasmic domains of CiTLR22(CiC)(ScE/CiC)were significantly up-regulated,and the IFN-? and IL-1? expression level in CIK cells overexpression ScE/CiC were significantly higher than that in CIK cells overexpressing CiTLR22(P<0.05),the virus titer were significantly reduced compared with that in CiTLR22 overexpressing cells.In addition,the study of the structural and functional characteristics of TLR22 gene in grass carp and barbel chub indicates that ScTLR22 and CiTLR22 participate in the immune responses of both barbel chub and grass carp against GCRV.The recognition region of ScTLR22 has a better ability to bind poly(I:C)than that of CiTLR22,and ScTLR22 can induce a stronger IFN immune response than CiTLR22 after GCRV stimulation.