Effects Of Dev Infection On NF-κB Signaling Pathway In Host Cell

Duck enteritis virus(DEV),Duck enteritis virus(Duck enteritis virus,DEV)is a threat to the healthy development of waterfowl breeding one of the important pathogens can cause ducks and geese and other water birds died.At present,the research on DEV has made great progress,but mainly in the viral genome and proteome,the pathogenesis is not yet fully understood.It is well known that the pathogenesis of a virus is essentially a process of interaction between a virus and a host,not only involving changes in molecules,cells and organs,but also in time and space.Nuclear factor kappa B(NF-κB)signaling pathway is an important way to affect the expression of various genes in animal level at the transcriptional level,which can play an important role in cell differentiation,immune response and inflammatory response.However,the role of NF-κB signaling pathway in DEV proliferation and its mechanism is unclear.Therefore,this study is based on the previous study,to further develop DEV infection on the host cell NF-κB signaling pathway of the study,to clarify the pathogenesis of DEV and NF-κB signaling pathway molecular mechanism research has some scientific significance.The main research contents include:1.The distribution of NF-κB gene in duck tissues after infected DEVThe tissue samples were collected from healthy ducks and DEV infected 96 h ducks.The specific primers were designed according to the nucleotide sequences of ducks NF-κB gene and GAPDH gene published in GenBank.GAPDH gene was used as internal reference,and real-time quantitative PCR(P0.05).Conclusion: The expression of NF-κB gene in healthy duck tissues was higher than that in the pancreas(9.0571 ± 3.8294),the spleen(1.0096 ±0.1648),the liver(0.8149 ±(0.7552 ± 0.0430),bursa of Fabricius(0.1797 ± 0.0696),lung(0.0436 ± 0.0016),heart(0.0354 ± 0.0054),thymus(0.5479 ± 0.0437),tracheal(0.3759 ± 0.0186)(0.0186 ± 0.0054),brain(0.0179 ± 0.0015)and muscle(0.0073 ±0.0022).The relative expression of NF-κB gene in DEV-infected duck was higher than that in pancreas(33.3750 ± 10.1016)(0.353 ± 0.072),thymus(0.7033 ± 0.1315),duodenum(0.4423 ± 0.1727),bursa of Fabricius(0.3354 ± 0.0780),trachea(0.2174 ± 0.0442),brain(0.1497 ± 0.1153)),Liver(0.1017 ± 0.0081),heart(0.0323 ± 0.0051),kidney(0.0278 ± 0.0047)and muscle(0.0033 ± 0.0007).The results showed that DEV infection could cause the transcriptional level of NF-κB gene in duck tissue to change in different degree,and the tissue with significant transcription level was pancreas,and the most obvious decrease was liver.How does these changes affect the pathogenesis of DEV pending further study.2.Effects of DEV infection on NF-κB signaling pathway in host cellAfter infection with DEV,the cells were collected at 1h,2h,4h,6h,12 h,24h,36 h,48h,60 h,72h,84 h,96h and 120 h after inoculation.RNA was detected by real-time fluorescence quantitative PCR.The supernatant was collected and the content of NF-κB signal pathway in the supernatant was detected by ELISA.Results: In DEV-infected cells,the NF-κB gene The levels of NF-κB,MyD88,IL-1β in the supernatant of DEV-infected cells were not significantly different from those in the normal control group(P <0.05)IL-8,IL-17,IL-32 and TNF-α secretion in different time groups showed varying degrees of irregular changes,in which NF-κB secretion in 4h ~ 12 h,36h ~ 72 h and 84 h ~ 120 h The secretion of MyD88,IL-17 and IL-32 increased at 84 h ~ 120 h,while the changes of IL-1β,IL-8 and TNF-α were irregular.The effect of these NF-κB signaling pathways on the proliferation of DEV has yet to be studied.3.Construction and identification of shRNA interference vector of NF-κB geneAccording to the sequence information of NF-κB gene published in GenBank,four different shRNA sequences were designed and inserted into pGPU6 / GFP / Neo vector.The positive recombinant plasmids were screened and sequenced.The positive recombinant plasmids could be digested with BamHⅠ,And the expected size of the two DNA fragments consistent;sequencing fragment size corresponding to 882 bp,928bp,997 bp and 1002 bp.These results indicated that the shRNA interference vector of NF-κB gene was successfully constructed in this study,and the basis for the study of DEV proliferation effect based on NF-κB gene was established.