Effect Of Knockdown Of HMGB1 Gene In Def Cells By CRISPR/Cas9 On Replication Of Duck Tembusu Virus

Duck tembusu virus(DTMUV)spreads rapidly in duck flocks,causing diseased ducks with mortality rates between 5% and 30%.The occurrence and epidemic of the disease has brought huge economic losses to the duck industry in China.HMGB1(high mobility group box protein 1,HMGB1)is a non-histone chromatin-related nuclear protein that has the ability to recognize,bind,and change the internal structure of DNA in the nucleus,and finally participates in DNA transcription,replication,and repair process.HMGB1 can promote the maturation and migration of DC cells by promoting dendritic cells to express cell markers such as CD40 and CD80.It also plays an important role in adaptive immunity.This study explores the editing of the HMGB1 gene in DEF cells by the CRISPR/Cas9 lentiviral system,and explores the interaction mechanism between the virus and the host's innate immunity.In this experiment,the CRISPR/Cas9 method was first used to knock down duck HMGB1(Duck HMGB1,duHMGB1)in duck embryo fibroblast(DEF)cells and infect DTMUV.For the first time,CRISPR/Cas9 The system's activity and the HMGB1 gene-mediated immune response mechanism of DTMUV infection were verified.Using duHMGB1 as the target gene,knock down the duHMGB1 gene in DEF cells and commissioned GK Gene to synthesize CAS9-custom-lentivirus(single vector)with the names LV-HMGB1-sgRNA,LV-HMGB1-sgRNA,and LV-HMGB1-sgRNA and negative control virus.Three pairs of CRISPR/Cas9 activity detection primers were synthesized.DEF cells infected with the above three lentiviruses and negative control viruses were used as templates to extract cellular RNA and reverse-transcribe them into cDNA.A PCR amplification reaction is performed based on the specific amplification primers.After the target bands were gel-recovered,the respective target bands were digested with mismatch enzymes,and the products were verified by 2% agarose gel electrophoresis.A CRISPR/Cas9 system was used to mediate DEF cell gene stabilization.Knock down method.DEF cells were used as a model to detect the virus replication in the cells after DTMUV infection.Quantitative PCR was used to detect IFN-?(IFN-?,IFN-?)and IFN-? in DEF cells infected with DTMUV(duHMGB1 knockdown).Changes in mRNA expression of ISGs(PKR,Mx,OAS).After DEF cells were infected with DTMUV,the expression levels of IFN-? and ISGs were significantly reduced,the expression levels of IFN-? were decreased by 12.4 times(P<0.001),and the expression levels of IFN-? were decreased by 8.1 times(P<0.01)The expression level of PKR was decreased by 14.2 times(P<0.001),the expression level of Mx was decreased by 7.6 times(P<0.01),and the expression level of OAS was decreased by 7.2 times(P<0.01).The results show that duHMGB1 can regulate the expression of IFN-?,IFN-?,PKR,Mx and OAS during DTMUV infection.This study successfully established a CRISPR/Cas9-mediated method for knocking down the HMGB1 gene in DEF cells.By in vitro infection with DTMUV,we verified the signaling pathway process involved by duHMGB1,and clarified the role of duHMGB1 in the host's innate immunity against DTMUV infection.The research results lay a theoretical foundation for the development of new drugs and vaccine adjuvants against DTMUV infection.