Study On The Role Of Duck HMGB2 In The Host's Innate Immunity Against Duck Plague Virus Infection

Ducks,geese and other waterfowls are the main breeding birds in China,accounting for75.0% of the world's total.However,duck virus disease has caused serious harm to duck industry in China and caused a series of public health safety problems.High Mobility Group box-2(HMGB2)is a highly conserved nuclear protein,which is widely expressed in Mammalian cells and plays an inflammatory role.HMGB2 is a highly viscous receptor for advanced glycation end products(RAGE)that binds to a variety of molecules on the cell surface.Hmgb2 plays a sentinel role in the innate immune response induced by viral nucleic acid,but if HMGB2 is not present in the cells,there is no uniform standard for the cells to know whether they have been infected by the virus.In view of this,this study is the first to systematically investigate the role of duck HMGB2(duHMGB2)in innate immunity against Duck Plague Virus infection:The first part is the amplification and sequence analysis of the duHMGB2 geneAccording to the predicted sequence of the duHMGB2 gene,the identification primers were designed,finally,the target fragment was amplified by specific primer(dhmgb2-f1 /R1).The results showed that the protein coding region(CDs)was composed of 624 bp,encoding 207 amino acids,and its molecular weight was about 24 KDA.According to the results of the phylogenetic tree,it was found that the duHMGB2 sequence cloned in this study had the highest homology with the original chicken,and had a distant relationship with mammals and reptiles.The second part is about the distribution of duHMGB2 in healthy duck tissuesThe distribution of duHMGB2 in healthy tissues of ducks was detected by fq-PCR.The spleen was used as the reference tissue and the Intron was-actin gene.The expression of HMGB2 in heart,liver,spleen,Lung,Kidney,brain,cerebellum,brain stem,thymus,pancreas,Bursa of Fabricius,Duodenum,Jejunum,Ileum,caecum,skin,muscle,glandular stomach,muscular stomach,trachea and esophagus were studied.The results showed that the expression of HMGB2 in Trachea,Jejunum,spleen,thymus,pancreas and skin was higher than that in trachea and Jejunum.The expression of HMGB2 in liver,kidney,Bursa of Fabricius,Ileum and Duodenum was weak.These results suggest that HMGB2 is widely distributed in ducks and may play a role in the innate immune response of the host.The third part is the role of HMGB2 in host innate antiviral immunityIn order to analyze the role of duck duHMGB2 in the innate immunity of the host against virus infection,the EUKARYOTIC expression plasmid of duHMGB2 and the Sirna interference plasmid were constructed,and the proliferation of the virus in different cells was compared,and analyze the expression of Pattern recognition receptor and cytokines in these cells to determine whether the DUHMGB2 has the ability to resist viral infection and viral signaling pathways,the results showed that:(1)the recombinant eukaryotic expression plasmid PCDNA3.0(+)-Duhmgb2-flag was successfully expressed in duck embryo fibroblast(DEF)cells,and most of the DUHMGB2 was localized in the nuclei of DEF cells after therecombinant plasmid was transfected into 48 hpi cells;(2)we transfected the DEF cells with PCDNA3.0(+)-duhmgb2-flag and reporter Plasmids(pGL3-NF-kB and pGL3-IRF7)and pRL-TK plasmids,harvested the cells 36 hours later and measured the luciferase activity,the results showed that the expression level of NF-kB in DEF cells transfected by duHMGB2 was higher than that in Control Cells(P<0.05).There was no significant difference in the expression level of IRF-7 between the two groups(P>0.05)(3)the levels of TLR2,IFN-A,IFN-?and IL-1? in DEF cells transfected with duHMGB2 were not significant(P<0.05),and the level of Rig-i in DEF cells transfected with duHMGB2 was lower than that in control cells(P>0.05)TLR3,TLR4,OAS,PKR,MX,IFN-?,TNF-? and IL-6 were significantly higher than those in Control Group(P<0.05).(4)the results of antiviral experiment showed that the replication ability of duck plague virus(DPV)was inhibited when the cells overexpressed Duhmgb2,and the replication ability of DPV was enhanced when the cells were weakened by RNA interference.The results showed that DUHMGB2 had the function of anti-DPV infection.The gene sequence of duHMGB2 was cloned and analyzed.The distribution of duHMGB2 in healthy duck tissues and the expression of duHMGB2 were detected,the role of PCDNA3 in anti-DPV innate immunity was further analyzed.These results lay a foundation for further study on the role of DHMGB2 in host innate immunity and provide a scientific basis for the screening of vaccine adjuvants.