Preliminary Stusies On The Role Of Cofilin On The Infection Process Of Duck Enteritis Virus

Duck enteritis virus?DEV?also called Duck plague virus?DPV?or Anatid herpesvirus 1?AHV-1?,has a wide range of organizations,is one of the important pathogens threaten and harming the healthy development of waterfowl breeding,but its mechanism of infection is not fully clear.Cofilin is an actin depolymerization factor in eukaryotic cells,participates in many biological processes,including the assembly of cytoskeleton,cell division,motility,adhesion and apoptosis.Studies have shown that remodeling of the actin cytoskeleton and on actin-filament severing by the regulatory protein cofilin,which can affect the infection of pathogens in host cells.However,no literature has been reported about the role of cofilin in the process of DEV infection and its mechanism.Therefore,based on this above discover,Transcription of cofilin was detected after DEV infection by fluorescence quantitative PCR?FQ-PCR?,then cofilin was used for further research on DEV multiplication by RNA interference technology,which provide the scientific basis for explanation of DEV pathogenesis.1.Cloning and Analysis and Establishment of fluorescence quantitative PCR method of Duck cofilin2 Gene.The specific primers were designed according to the gullas cofilin2 gene?NM₀01004406.1?in Gen Bank,and amplified for the duck cofilin2 gene,then this gene characteristic was analyzed using bioinformatic softwares.The specific primers were designed according to duck cofilin2 gene sequence,established the fluorescence quantitative PCR for cofilin2.The results showed that the length of cofilin2 gene was 498 bp in Sansui duck,which encode 166 amino acids.The identities of Cofilin2 gene in Sansui duck were 94.4%,91.6%,91.6%,87.6%,77.7%,70.5% and 69.5% shared with that of Gallus,Macaca mulatta,Bos taurus,Mus musculus,Salmo salar,Homo sapiens and Pig,respectively.The phylogenetic tree analysis result showed that Cofilin2 gene maintained a highly conservative among different species.The secondary sructure of this encoding protein was characteristical with random curl,alphahelix and extended strand region,and the curved spiral in tertiary structure.The recombinanted plasmid of cofilin2 gene has been used as positive sample was detected by Fluorescence quantitative PCR,The results showed that:There is good linear relationship between the Ct value and the number of gene copies,the linear relationship of standard curve was Y=-3.31x+38.29;The specificity FQ-PCR method was established with detectable amount of 1.0 x 102copies/ L.2.Change of transcription for Cofilin2 gene after DEV infection in duck tissues.After picking healthy Sansui duck and infecting DEV artificially,duck samples were collected at different time to extract total RNA from the sample.Using the established real-time fluorescent quantitative PCR method to detect the transcription level of tissue cofilin2 gene.The transcriptional results of the cofilin2 gene in the normal and DEV-infected groups at the same time point showed that the transcription of cofilin2 was highest in leg muscles,followed by heart,and lowest in spleen,bursa of Fabricius and thymus in the normal group;The transcription of cofilin2 was highest in the heart,followed by the leg muscle,and lowest in the spleen,bursa of Fabricius and thymus in the DEV-infected group.At the same time,the transcription of cofilin2 gene in DEV-infected groups did not show any regularity comparing with the normal group in different tissues.The transcription level of cofilin2 in the infection group was higher than in the normal group in the heart,which most significant at 72hours;Except for 24 h,the transcription of cofilin2 was higher in the infected group than in the normal group in the liver,and the most significant was at 48h;Except for 48 h,the transcription of cofilin2 in the infected group was higher than in the normal group in the spleen,and the most significant was at 72 h;Except 72 h,the transcription of cofilin2 was higher in the infected group than in the normal group in the lungs,and the most significant was at 24h;In the kidney,the transcription of cofilin2 group was higher in the infected than in the normal group at 24 h and 48 h,and was lower in the infection than in the normal group at 72 h and 96h;In brain tissue,the transcription of cofilin2 was lower in the infection group than in the normal group at 24 hours,higher than in the normal group at 48 hours and 72 hours,and lower than in the normal group at 96 hours;In thymus,the transcription level of cofilin2 was higher in the infected group than that in the normal group at different times,and the most significant was at 96 hours;In the duodenum,the transcription level of cofilin2 was higher in the infected group than in the normal group at 24 h and 48 h,and lower than in the normal group at 72 h and 96h;In bursa of Fabricius,the transcription of cofilin2 was higher in the infected group than in the normal group from 24 h to 72 h,and was lower than in the normal group at 96 h.In leg muscle,the transcription of cofilin2 in the infected group was lower than in the normal group at different time points,of which the most significant was at48 hours.Thus,the transcription of cofilin 2 gene in heart and thymus of DEV-infected group was higher than that of normal group at each time point,and the transcription of cofilin 2 in leg muscle of DEV-infected group was lower than that of normal group at each time point.Transcription of cofilin2 did not show some regularity in other tissues with DEV infection.3.Effect of Cofilin2 Gene RNAi on DEV Replication ProcessThe interference plasmids cofilin-shRNA-86,cofilin-shRNA-247,cofilin-shRNA-304,and cofilin-sh RNA-451 were designed and constructed based on the above described duck cofilin2 gene sequence?Gen Bank serial number MF434775?.Duck Embryo Fibroblast?DEF?were transfected with the above interference plasmids,and then the interference plasmid with the highest inhibition efficiency was selected by fluorescence microscopy and FQ-PCR.The highest efficient plasmids was used to inhibit the cofilin2 gene of DEF,and then the effect of Coliin2 gene RNAi on DEV replication process was analyzed by FQ-PCR method.The results showed that The interference plasmids were successfully transfected and expressed by fluorescence microscopy in DEF;The FQ-PCR method confirmed that the silencing efficiency of the four interference plasmids respectively was 54.02%,17.55%,41.12%,and4.96%.Among them,the silencing efficiency of cofilin-sh RNA-86 was the highest;The silencing efficiency was 42.59%-56.23% at 48 h,72h,96 h and 120 h after transfection of cofilin-sh RNA-86 in DEF;DEF infected with DEV.after 24 h of cofilin-shrna-86 transfection,the DEV nucleic acid amount was increased in the cofilin-sh RNA-86 infection gruop compared with the normal group at different time points.In summary,the duckling cofilin2 gene sequence was successfully sequenced in this experiment;he FQ-PCR detection method for cofilin2 gene was Established;The transcription of cofilin2 gene in normal group and DEV-infected group showed similar trends in different samples at the same time point in ducks;The transcriptional changes of the cofilin 2 gene in different periods of the same tissue do not show some regularity;Cofilin-sh RNA-86 with obvious silencing effect was selected by filtration;By interfering with the host cell cofilin2 gene expression,DEV proliferation can be significantly promoted.