Expression Characteristics And Immune Regulatory Mechanisms Of Toll-Like Receptor 20.2 Of Grass Carp(Ctenopharyngodon Idellus) In Response To GCRV

Grass carp hemorrhagic disease has seriously threatened the grass carp cultivation industry.It was proved that maternal immunized with GCRV attenuated vaccine grass carp offsprings had more resistance to GCRV than those of common grass carps.In this study,maternal immunized and common grass carp eggs were used to screen the key factors in the immune regulation of GCRV by proteomics study.The expression patterns and immune regulation mechanism of the key factor in response to GCRV were explored with methods of molecular and cell biology.The main results were as follows:1.Differentially expressed proteins were screened in eggs of maternal immunized and common grass carpBased on iTRAQ technique,138 differentially expressed proteins(DEPs)were identified in eggs of maternal immunized three concentrations of GCRV attenuated vaccine and common grass carps,including 67 up-regulated proteins and 71 down-regulated proteins.Gene Ontology analysis showed that DEPs were mainly enriched in immune response,DNA double-strand breaking and autophagy processes.KEGG pathway analysis showed that DEPs were mainly enriched in TLR and m TOR signal pathways.The TLR20.2 was found in maternal immunized groups compared with the control group as a significant DEP with a maximum fold change,10.4 times of the control group.Combined with the transcriptome data of grass carp and barbel chub infected by GCRV,it was shown that TLR20.2 played an important role in regulating the immune response to GCRV infection.2.Maternal immunization improved the expression of grass carp TLR20.2 during early embryonic developmentqPCR,and ELISA were used to determine the expression patterns of TLR20.2 in early embryonic development period of maternal immunized and common grass carps.The m RNA expressions of TLR20.2 gene in the embryonic development period displayed a trend of first decreasing and then increasing.The lowest expression level of TLR20.2 in maternal immunized grass carp was found in the two-cell stage,which was 3% of that in the egg stage.The m RNA expression levels of TLR20.2 was higher than those of the control group except for the two-cell stage and gastrula stage,and there are significant differences in the neurula stage,somite formed stage and eye lens formed stage between maternal immunized and control groups.The protein expression levels of TLR20.2 were decreased in the maternal immunized group during the embryonic development period.The lowest expression was shown in the eye lens formed stage which was 83% of that in the egg stage.Except for somite formed stage,the protein expression of TLR20.2 were higher in the maternal immunized group than those in the control group and there were significant differences in egg,fertilized egg,blastocyst and gastrula stage.All the results above showed that maternal immunization up-regulated the expressions of grass carp TLR20.2 during early embryonic development and the expressions of TLR20.2 in maternal immunized grass carp were gradually decreased during embryonic development.3.TLR20.2 was involved in the immune response of grass carp against GCRV infectionThe expression levels of grass carp of TLR20.2 were detected by ELISA,qPCR and Western blot.The TLR20.2 protein expressions of healthy grass carp was detected by ELISA and it was highest expressed in the brain and the lowest in kidney.The m RNA expression level of TLR20.2 in liver and head kidney of grass carp firstly increased then decreased,and reached the expression peaks at 120 h and 72 h after GCRV stimulation which were extremely significant higher than those of the control group with 12.2 and 5.8 fold,respectively.The expression pattern of TLR20.2 in spleen was gradually up-regulated,and there was a significant difference at 168 h after GCRV stimulation,which was 10.2 times higher than that of the control group.In vitro,subcellular localization result revealed that TLR20.2 was expressed in both nucleus and cytoplasm of CIK cells.Then,TLR20.2-overexpressing CIK cells were infected with GCRV,the expression levels of GCRV outer capsid protein VP2 and VP7 genes were significantly down-regulated by 348 and 249 times,respectively.The expression levels of TLR20.2 protein in TLR20.2overexpressed cells stimulated with GCRV were higher than those in the control group,and the significant difference was observed at 48 h post GCRV stimulation.The results suggested that TLR20.2 was involved in the immune response of grass carp against GCRV infection.4.Grass carp TLR20.2 was regulated by HMGB1 and participated in GCRV immune regulation through HSP90/TRIM14The transcription factor HMGB1 that binded TLR20.2 promoter was screened by DNA pull down and TLR20.2 interacting with HSP90 and TRIM14 were proved by co-immunoprecipitation and bimolecular fluorescence complementation.Immunofluorescence results showed that the expression distribution of HSP90 in liver and muscle of grass carp stimulated with GCRV gradually increased,and the strongest fluorescence signals were shown in 7th and 3rd day post GCRV infection,respectively.The expression distribution of TRIM14 increased gradually in the liver and muscle of grass carp infected with GCRV,and the TRIM14 signal overlapping with VP7 signal were obviously observed and the the strongest signal was detected until 7th day post GCRV stimulation.The m RNA expression levels of IRF7,IFN1,VP2 and VP7 genes presented an increased trend in the overexpressing HSP90 CIK cells after GCRV infection.The expression of TRIM14,IRF7,IFN1,VP2 and VP7 genes increased in overexpressed TRIM14 CIK cells post GCRV stimulation.These results suggested that HMGB1 could bind TLR20.2 promoter.TLR20.2interacted with HSP90/TRIM14 and activated IFN signaling through IRF7 to mediate the immune response of GCRV infection.The results suggested that HMGB1 could combine with TLR20.2 promoter;TLR20.2 participated in the IFN pathway in response to GCRV infection through HSP90/TRIM14.In summary,the expression properties and regulation mechanism of immune responses to GCRV infection were investigated in this study.These results laid a foundation for screening the anti-GCRV immune factors and provided a basis for analyzing the mechanism of immune regulation in response to GCRV infection.