Study On The Mechanism Of Anti-duck Tembusu Virus Effect Of Duck IRF1 And Its Application

Duck and other waterfowls are the main farmed poultry in China,and the breeding amo unt accounts for about 70% of the world total.In recent years,duck tembusu virus(DTMU V)has seriously harmed the duck breeding industry in our country.The duck tembusu virus disease caused by DTMUV mainly causes egg production of laying ducks decline and slow growth of meat ducks.The disease spreads rapidly and has become a serious disease that hin ders the healthy development of duck industry in our country.From June to December,2019,our laboratory obtained 124 samples of sick ducks from various farms in Shandong.The d etection and analysis results showed that the detection rate of DTMUV was about 14.5%,an d most of the sick ducks were about 15 days old.Interferon regulatory factor 1(IRF1)was first discovered in 1988.It can regulate the ex pression of IFN-β and is a very important transcription factor.The antiviral effect of IRF1 ha s been deeply studied in mammals and other higher animals,but less in poultry,especially w aterfowl.The laboratory was first acquired the gene sequence of the duck IRF1,and confirm ed its participation in the host antiviral innate immunity,but the exact mechanism was unkno wn In order to further clarify the antiviral mechanism and clinical application of Cherry Vall ey duck IRF1,the following four aspects have been done in this study:Part one: the effect of knockdown of IRF1 gene of Duck embryo fibroblast(DEF)cells on DTMUV replication and detection of related immune factors.In this experiment,three interference RNAs of du IRF1 were designed: Si-du IRF-1,Si-d u IRF1-2 and Si-du IRF1-3.The detection results showed that the interference efficiency of Si-du IRF1-2 was the highest,so Si-du IRF1-2 was selected for the subsequent interference exp eriment.Si-du IRF1-2 and Si-NC were transfected into DEF cells,respectively,and DTMUV infection experiment was carried out at 36 h after transfection,and cells were collected at 12 hpi,24hpi,36 hpi and 48 hpi,respectively.The changes of DTMUV viral load and the expres sion of several major pattern recognition receptors,interleukin,interferon,antiviral protein a nd major histocompatibility complex in the experimental and control group were detected by fluorescent quantitative PCR.The results showed that the viral load of the experimental gro up was higher than that of the control group at four time points.In addition,compared with t he control group,the expression of immune factors in the experimental group almost showed a downward trend.For example,TLR4 decreased 3 times at 12hpi(P < 0.001),IL-1β decrea sed 3 times at 36hpi(P < 0.001),and PKR decreased 5.8 times at 48hpi(P < 0.001).Theabo ve results indicated that knocking down the expression of duck IRF1 would promote the repl ication of DTMUV.The reason for this phenomenon might be that the expression of partial downstream immune factors of IRF1 was down-regulated after the expression of IRF1 was s uppressed.Part two: construction of prokaryotic expression vector of Cherry Valley duck IRF1.In this experiment,RNA was extracted from the spleen of healthy Cherry Valley duck a nd reverse transcribed into c DNA,which was used as a template for amplifying IRF1 gene.According to the predicted sequence of duck IRF1 published on NCBI,primers were designe d for PCR reaction.The PCR products were electrophoresed and the gel of the target fragme nt was recycled for sequencing.Finally,the target gene IRF1 was obtained.The prokaryotic expression vector p ET32a(+)-His was double digested,and the obtained target fragment was homologously recombined with the linearized vector,and the recombinant products were tra nsformed into DH5α competent cells.A single smooth colony was selected for bacterial liqui d PCR identification and sent for sequencing.SDS-PAGE and Western Blot were further use d to confirm that the recombinant plasmid p ET32a(+)-du IRF1-His could be successfully exp ressed.Part three: preparation of polyclonal antibody against Cherry Valley duck IRF1 and its application.In this experiment,the expression conditions of the recombinant protein were explored,and the results showed that the maximum expression of the recombinant protein was obtaine d under the conditions of inducer IPTG final concentration of 1.0 mmol/L,induction tempera ture of 30℃ and induction time of 6 hours.Through the verification of inclusion body protei n,it could be seen that the protein was expressed in the form of inclusion body.Purified by n ickel column,the concentrations of No.4 and No.5 protein eluents were higher,which could be used for subsequent experiments.The purified protein was mixed with Freund’s adjuvant i n equal volume,and female BALB/c mice aged 4-6 weeks were injected subcutaneously into the back at multiple points after complete emulsification,and each mouse was immunized w ith 100 μg purified recombinant protein.10 days after the three immunizations,blood was co llected and the serum was separated,then the positive serum was purified by antigen affinity chromatographyand.The titer of the prepared polyclonal antibody was determined by indirec t ELISA to reach 1:32000,and Western Blot was proved that the polyclonal antibody had go od specificity.The polyclonal antibody of duck IRF1 prepared in this experiment was very i mportant for the innate immune system of ducks.It could detect the expression changes of d uck IRF1 during the antiviral process,which had very important application value.In this ex periment,purified duck IRF1 protein with final concentration of 1 μg/m L was added into DE F cells of experimental group and control group,after incubation for 12 hours,DTMUV was used to infect experimental group,while control group was not treated.The cells were collec ted at 24 hpi,and the prepared duck IRF1 polyclonal antibody was used as the primary antib ody for Western Blot to detect the expression of IRF1 protein.The results showed that there was no significant difference in IRF1 protein expression between the two groups.We specul ated that duck IRF1 protein might play an antiviral role through phosphorylation and other p athways.Part four: recombinant protein of Cherry Valley duck IRF1 in prokaryotic expression ha d the effect of resisting DTMUV infection.In this experiment,DEF cells added with duck IRF1 recombinant protein with final con centration of 1 μg/m L were used as experimental group,while the control group was not trea ted.After incubation for 12 hours,two groups cells were infected with DTMUV,and the cell s were collected at 12 hpi,24 hpi and 36 hpi for fluorescence quantitative PCR reaction to de tect the DTMUV virus load.The results showed that the DTMUV virus load in the experime ntal group with duck IRF1 recombinant protein was lower than that in the control group at th ree time points.The above results indicated that exogenous expressed recombinant duck IRF1 protein had the function of anti-DTMUV infection,which provided the possibility for the p reparation of new anti-DTMUV drugs.In this study,RNA interference experiments showed that knocking down IRF1 gene in DEF cells could promote the replication of DTMUV,and down-regulate the expression of se veral major pattern recognition receptors,interleukin,interferon,antiviral protein and major histocompatibility complex in the early stage of DTMUV infection,which proved that duck IRF1 participated in the anti-DTMUV innate immune response.Because duck IRF1 can inhi bit DTMUV replication,we hope to explore the clinical significance of duck IRF1 by prepari ng duck IRF1 polyclonal antibody and studying the clinical application of recombinant prote in of duck IRF1 in prokaryotic expression.In this experiment,we constructed the prokaryoti c expression recombinant plasmid p ET32a(+)-du IRF1-His,and further prepared duck IRF1 p olyclonal antibody,which provideed a detection method for the research of IRF1 in innate i mmunity in our laboratory and in this field.Finally,it was found that DEF cells added into d uck IRF1 prokaryotic expression recombinant protein could inhibit the replication of DTMU V in the early stage of DTMUV infection,which provided a possibility for developing new a ntiviral drugs against DTMUV.