Targeting NF-?B1 Gene RNAi Effects On DEV Proliferation

Duck enteritis virus(DEV)is one of the important pathogens that threaten and endanger the healthy development of the waterfowl breeding industry,but its pathogenesis has not been fully elucidated.NF-?B molecule is a key molecule in the NF-?B signaling pathway of animal organisms and plays an important role in many biological processes of animals,associated with inflammatory processes and viral infections,and the key regulators of NF-?B signaling pathway are cytokines such as IL-1?,no studies have reported associations between DEV infection and NF-?B signaling pathways.This study used RNAi interference technology to silence NF-? B1 gene,analysis of the effect of NF-? B1 gene on DEV proliferation,and analysis of changes in IL-1 ?,a key regulator of NF-? B signaling pathway,provide some basis for revealing the pathogenesis of DEV.1.Establishment of RT-PCR detection method for duck source IL-1? geneDesign specific primers based on duck source IL-1? gene in GenBank,and PCR amplification to obtain IL-1? gene fragment,recovered and purified and ligated to pMD19-T vector,then transformed into E.coli and enriched,extracting recombinant plasmid for identification,SYBR Green I real-time PCR was performed using recombinant plasmid as template,and conduct specificity,repeatability and sensitivity testing.The results show:The established duck-derived IL-1 ? gene fluorescence quantitative PCR method has a good amplification curve,the standard curve equation y =-3.329 x + 39.731,the amplification efficiency is 99.7%,and the correlation coefficient is 1,The intra-assay repeat coefficient of variation detected by the real-time PCR is less than 2%,and the inter-assay repeat coefficient of variation is less than 3%;the detection sensitivity of this method is 1.74×101copies·?L-1,which is 100 times that of the common PCR method,the melting curve has no peaks,indicating good specificity.These results indicate that the SYBR Green I real-time PCR method for duck-derived IL-1? gene was successfully established in this study.2.Analysis of transcriptional changes of IL-1? gene in duck tissue organs infected with DEVSixty healthy 40 d healthy ducks were randomly divided into two groups,the infectiongroup and the normal group,the infected group was intramuscularly inoculated with DEV and the normal group was injected with normal saline,experimental ducks were killed at 12 h,24h,36 h,48h,60 h,72h,84 h and 96 h after treatment,collect heart,liver,spleen,lung,kidney,brain,duodenum,trachea,muscle,bursa,thymus and pancreas,and extract tissue DNA and mRNA samples,identification of DEV content by the common PCR method of DEV NP gene established in the early stage of the laboratory,detection of IL-1?1 gene transcription level by RT-PCR.The results show:The DEV NP gene was not detected in the tissues and organs of the infected group,indicating that the DEV infection was successful.The IL-1 ? gene transcription level in the infected organs of ducks in the infected group increased at different time points and was higher than that in the normal group.At 72 h,the IL-1? transcription level of lung,spleen and kidney was up-regulated most,reaching 100 times of the normal group.These results indicate that DEV infection significantly up-regulates IL-1 ?transcription levels.3.Effect of targeting NF-kB1 gene RNAi on DEV proliferation and secretion of major cytokinesThe recombinant plasmid pGPU6/GFP/Neo-NF-kB1-1~4 constructed in the previous stage of the laboratory was transfected into DEF cells for silencing analysis and the effect of recombinant plasmid on cell proliferation was detected.Virus infection and plasmid transfection were performed in three ways to treat DEF cells,ie,virus infection for 24 h followed by plasmid transfection,plasmid transfection for 24 h,virus infection,viral infection and plasmid transfection,transfection fluorescence was observed at 36 h,48h,60 h and 72 h after treatment.At the same time,the transcription levels of DEV-NP gene and IL-1? gene were detected by RT-PCR.The secretion of IL-1?,myD88,TNF?,IL-6 and IL-8 in the downstream of NF-?B signaling pathway was detected by ELISA.The results showed that the interference efficiency of recombinant plasmid pGPU6/GFP/Neo-NF-kB1-2 was 78.77%,and the silencing effect was the best.The interference of recombinant plasmid on the survival of DEF cells was not significant.In the three treatment groups,DEV experiment Compared with the group,the interference effect of the IFN group on the DEV proliferation was not significant,but the first interference after the poisoning group and the simultaneous poisoninginterference group all significantly inhibited the proliferation of DEV,and the 48 th hour was the simultaneous poisoning interference group.The silencing effect was the best,the silencing efficiency was 83%,and the silencing effect was the best in the 72 h after interfering with the toxic group,and the silencing efficiency was 77%.The IL-1? transcript level increased first and then decreased in the DEV infected group.In the RNAi interference group,the increase was first and then increased,while in the RNAi interference + DEV infection group,the expression level of NF-?B downstream factor showed a certain change in different RNAi treatment groups.The expression of-1? was similar to that in the infection group and the interference and infection groups,and all of them were lower than the normal group at 36 h,48h and 60 h in the interference control plasmid control group,and 36 h,60h and 72 h in the DEV control group.Higher than the normal group,at Interference recombinant plasmid +DEV group presented repeated changes of increased and then decreased.These results indicate that targeting RNA interference of NF-kB1 gene can inhibit DEV proliferation and affect the secretion of downstream factors of NF-?B.In conclusion,this study successfully established a duck-derived IL-1? gene fluorescent quantitative PCR method;DEV infection can lead to up-regulation of IL-1? gene transcription level in host cells;targeting NF-kB1 gene RNAi interference can inhibit DEV proliferation Affects the secretion of factors downstream of the NF-?B signaling pathway.