Effect Of REV Infection On Proteomics And Endoplasmic Reticulum Stress Signaling Pathway In Bursa Of Fabricius Of SPF Chickens

In order to further explore the pathogenic mechanism of REV infection host,fill the gaps in the study of proteomics and endoplasmic reticulum stress signaling pathways in bursa of fabrics after REV-infected chickens,and lay an important theoretical foundation for the prevention and treatment of REV.In this study,one-day-old SPF chickens were used as the research object.On the basis of successful establishment of SPF chickens infected with REV,TMT labeling proteomics technology was used to detect the changes of chicken bursa of Fabricius protein before and after REV infection at 7,14,21 and 28 days.Bioinformatics methods were used to classify the differential expression proteins obtained by proteomics with GO secondary annotation,KEGG pathway,protein domain enrichment analysis and pattern expression cluster analysis.Meanwhile,ELISA and RTq PCR were used to detect the ER stress signaling pathway related molecules in the bursa of Fabricius and apoptosis-related molecules induced by ER stress in SPF chickens 7,14,21 and 28 days after REV infection.The pathological changes of tissue morphology and ultrastructure of bursa of Fabricius in SPF chickens infected with REV were observed by traditional histopathological methods and electron microscopy.This project provides scientific experiments and theoretical basis for further study of the immunosuppressive mechanism caused by REV.The main research results are as follows:(1)Establishment of disease model of SPF chickens infected with REVIn this study,one-day-old SPF chickens were infected with RE virus solution by intraperitoneal injection.The PCR method was used to detect the conservative fragment LTR of REV in the bursa of Fabricius of infected chickens.The results showed that after 1 % agarose gel electrophoresis of PCR products,275 bp specific products were observed in the electrophoresis channel of infected chickens under ultraviolet light,while no specific band was found in the corresponding electrophoresis channel of control chickens,indicating that the disease model of REV infected SPF chickens was successfully established.It laid the foundation for the further study of the project.(2)Effect of REV infection on proteomics of SPF chicken bursa of Fabricius1)Analysis of differentially expressed proteins in bursa of Fabricius of SPF chickens infected with REVBy mass spectrometry analysis,compared with the control group,1127 differentially expressed proteins were identified on the 7 th day after REV infection in SPF chickens,including 300 upregulated proteins and 827 down-regulated proteins.A total of 999 differentially expressed proteins were identified on the 14 th day after REV infection in SPF chickens,of which 265 were up-regulated and 734 were down-regulated.On the 21 st day after REV infection in SPF chickens,a total of 910 differentially expressed proteins were identified,including 231 up-regulated proteins and 679 down-regulated proteins.On the 28 th day after REV infection,a total of 1138 differentially expressed proteins were identified,including 338 up-regulated proteins and 800 downregulated proteins.2)Functional enrichment analysis of differentially expressed proteins in bursa of Fabricius of SPF chickens infected with REVEnrichment analysis results show that compared with the control chickens without REV infection,on the 7th day after REV infection,the differentially expressed proteins were mainly enriched in nucleotide metabolism,transmembrane transport regulation,neutrophil activation and neutrophil-mediated immunity in the biological process.In cell components,the differentially expressed proteins are mainly enriched in membrane protein complexes,mitochondrial membrane,organelle intima and respiratory chain complexes.In molecular function,the differentially expressed proteins are mainly enriched in nucleotide binding,oxidoreductase activity,NADH dehydrogenase activity,etc.On the 14 th day after REV infection in SPF chickens,the differentially expressed proteins were mainly enriched in nucleotide metabolism,nucleotide biosynthesis,pathogen defense,neutrophil activation and neutrophil-mediated immunity in the biological process.In cellular components,the differentially expressed proteins are mainly concentrated in organelle envelope,membrane protein complexes,mitochondrial membrane,vesicles,respiratory chain complexes and chaperones.In molecular function,the differentially expressed proteins are mainly enriched in nucleotide binding and oxidoreductase activity.On the 21 th day after REV infection in SPF chickens,the differentially expressed proteins were mainly enriched in nucleotide metabolism,ion transport and B-cell mediated immunity in the biological process.In cell components,the differentially expressed proteins are mainly enriched in mitochondrial envelope,secretory vesicles,membrane protein complexes,respiratory chain complexes and proteasome regulatory particles.In molecular function,the differentially expressed proteins are mainly enriched in nucleotide binding,lipid binding,oxidoreductase activity and chaperone.On the 28 th day after REV infection in SPF chickens,the differentially expressed proteins were mainly enriched in nucleotide metabolism,ion transport,neutrophil activation,actin cytoskeleton regulation and immunoglobulin-mediated immune response.In cell components,differentially expressed proteins are mainly enriched in secretory vesicles,cell connections,membrane protein complexes,supramolecular polymers,and respiratory chain complexes.In molecular function,the differentially expressed proteins are mainly enriched in protein complex binding,phospholipid binding,oxidoreductase activity,G protein complex binding and proton transmembrane transporter activity.3)Cluster analysis of differentially expressed proteins in bursa of Fabricius of SPF chickens infected with REVCluster analysis of pattern expression showed that in the first cluster,subcellular components migration,actin cytoskeleton organization,supramolecular complexes,cytoskeleton,structural molecular activity,biological processes and molecular functions,as well as immunoglobulin I domain clustering of differentially expressed proteins,showed a significant decrease in the expression level 7 days after REV infection in SPF chickens,and showed a stable trend from 7 to28 days.In the second cluster,the differentially expressed proteins in the intestinal immune network,calcium signaling pathway,NF-?B immune-related signaling pathways,lymphocyte-mediated immunity and other biological processes were generated,showing an increase in expression after REV infection in SPF chickens.In the third cluster,the differentially expressed proteins in intracellular protein transport,mitochondrial membrane and other biological processes,as well as G protein-coupled receptor binding,GTPase activity and other molecular functions were significantly decreased within 7 days after REV infection in SPF chickens,but steadily increased between 7 and 21 days.In the fourth cluster,the related biological processes and protein domains were manifested in protein digestion and absorption,endoplasmic reticulum,globulin,etc.,and the expression levels of differentially expressed proteins were significantly decreased first and then slowly increased within 21 days after REV infection in SPF chickens.In the fifth cluster,the differentially expressed proteins in the intestinal immune network pathway and adaptive immune response of antigen treatment and presentation,lymphocyte-mediated immune and other biological processes,as well as the molecular functions of immunoglobulin receptor activity and transmembrane signal receptor activity were significantly increased within 21 days after REV infection in SPF chickens,and slightly decreased from 21 to 28 days.The differentially expressed proteins in cluster 6,including the differentially expressed proteins in platelet activation,purine metabolism,ligase activity and other pathways or molecular functions,showed a continuous decline within 28 days after REV infection in SPF chickens.(3)Effect of REV infection on endoplasmic reticulum stress signaling pathway in SPF chickens1)REV infection induced endoplasmic reticulum stress in bursa of Fabricius of SPF chickensThe content of glucose regulatory protein-78(GRP-78)in bursa of Fabricius of SPF chickens infected with REV was detected by ELISA.The results showed that compared with the control chickens,the content of GRP-78 in bursa of Fabricius of SPF chickens infected with REV was significantly increased on the 7th,14 th and 21 th day,indicating that REV infection can induce endoplasmic reticulum stress in bursa of Fabricius of SPF chickens.2)Identification of endoplasmic reticulum stress signaling pathway activated by REV infection in SPF chickensThe unfolded protein response(UPR)pathway specifically activated in SPF chickens infected with REV was identified by RT-q PCR and ELISA,respectively.The results showed that the protein contents of phosphorylated protein kinase-like endoplasmic reticulum kinase(p-PERK),phosphorylated eukaryotic promoter(p-eIF-2?)and activating transcription factor-4(ATF-4)in the bursa of Fabricius of SPF chickens infected with REV were higher than those of the control chickens in varying degrees,indicating that REV infection could activate the protein kinase-like endoplasmic reticulum kinase(PERK)pathway in the bursa of Fabricius of SPF chickens.ELISA results showed that phosphorylated inositol requiring enzyme-1(p-IRE1)and transcription activator-6(ATF-6)in bursa of Fabricius of SPF chickens were not significantly increased at each time point after REV infection,and the m RNA expression levels of X-box binding protein-1(Xbp-1),EDEM,CANX and CALR were not significantly increased.The above results showed that REV infection could activate the PERK pathway of unfolded protein response in bursa of Fabricius of SPF chickens,but did not activate the IRE-1 and ATF-6 pathways.3)Apoptosis of bursa of Fabricius of SPF chickens induced by ER stress after REV infectionELISA and RT-q PCR were used to detect the expression of CHOP protein and Bcl-2 and Caspase-3 m RNA in bursa of Fabricius of SPF chickens infected with REV at 7,14,21 and 28 days after infection.The results showed that the content of CHOP in bursa of Fabricius of SPF chickens infected with REV was higher than that of control chickens at different time points,especially at 14 and 21 days(P < 0.05).Bcl-2 m RNA expression decreased to varying degrees after REV infection in SPF chickens,and it was significantly decreased on the 14 th and 28 th days(P < 0.05).The expression of Caspase-3 m RNA was increased at 7–21 days after REV infection in SPF chickens,and was significantly increased at 7 and 21 days(P < 0.05).The above results showed that REV infection in SPF chickens induced apoptosis of bursal cells through the PERK-eIF-2?-ATF-4-CHOP pathway.(4)Changes in pathological morphology and ultrastructure of bursa of Fabricius in SPF chickens infected with REVCompared with the control chickens without REV infection,under the optical microscope,it can be seen that the reticular endothelial tissue in the bursa of Fabricius of SPF chickens infected with REV proliferated,the lymphocytes in the bursa of Fabricius cortex decreased,and the lymphoid follicles in the bursa of Fabricius decreased.Further electron microscope observation showed apoptosis,swelling of mitochondria and endoplasmic reticulum,and focal necrosis in bursa of Fabricius.The results indicated that REV infection could result in structural damage of bursa of Fabricius in SPF chickens.In conclusion,the protein expression profile of bursa of Fabricius changed significantly after REV infection in SPF chickens,and REV infection can lead to endoplasmic reticulum stress in bursa of Fabricius and activate the PERK pathway of UPR to further induce apoptosis,which provides a scientific experiment and theoretical basis for further study of the pathogenesis of REV and the immune response to the host.