Identification,Biological Characteristic And Antiviral Function Of Goose Tripartite Motifcontaining Protein 25 In Goose

TRIMs play a critical role in many biological progresses,such as cell diff erentiation,apoptosis,transcription regulation and cellular signaling.As one of the ISGs,TRIM25 ubiquitin RIG-I in viral infection.Goose TRIM25 was clon ed from gosling lung and sent for sequencing.The antiviral function and effect of TRIM25 on IFNs and ISGs were detected.1.Clone of goose TRIM25 and molecular biological characteristic of goose TIR M25The cloned 1892 bp cDNA of TRIM25?KX₃64385?with a 1662 bp con served ORF coded a predicted 554 amino acid protein,which contains an N-te rminal BCC motif:a B-box domain and a Coil-coil domain,while short of 390 bp N-terminal sequence compared with duck TRIM25,which contains a RIN G domain and partial B-Box1 domain.We carried out a series of bioinformatic s analysis with the cloned goose TRIM25 sequence and other TRIM25 sequenc es from different species which were submitted to GenBank.The phylogenetic tree analysis showed that:goose TRIM25 is closer to duck?Anas platyrhyncho s?TRIM25 and chicken?Gallus gallus?TRIM25,which is in line with the ho mology analysis.We also found that almost all the TRIM25 proteins contain th e zinc-finger domain sequence:CX2CX?9-39?CX?1-3?HX?2-3?C/HX2CX?4-48?CX2C,which is the E3-ligase classic domain.2.The tissue expression profiles and immunological characterization of goose TRIM25We detected the transcription level of TRIM25 in vivo,qPCR result show ed that TRIM25 was highly expressed in the blood,thymus,caecum tonsil and harderian gland.In addition,TRIM25 was highly expressed in bursa of Fabrici us in gosling while lowly expressed in 3 months old goose.To determine whe ther TRIM25 could be induced by IFNs,virus and TLRs agonists,we detected the TRIM25 transcription level in GEFs and PBMCs treated with GPV,H9N2,IFN?,R848,Poly?I:C?or ODN 2006.The result indicated that all the agonist s,IFN?and virus treatment could significantly up-regulate the transcription of goose TRIM25.3.The effect of TRIM25 in inhibiting of viral repilcationTo determine the effect of TRIM25 to the profileration of DPV,DEFs wer e transfected with GoTRIM25 and DuRING-GoTRIM25,and challenged the cel ls at 24 hours post transfection with Duck plague virus?BAC-DPV-GEFP?(TCI D50:10^-6.125/100?L),and the fluorescent images were collected at 24 and 48hours post infection.The result indicated that the fusion protein DuR-GoTRI M25 could significantly inhibit DPV compare with GoTRIM25.To determine t he effect of TRIM25 to the profileration of NDV,DEFs were transfected with GoTRIM25 and DuRING-GoTRIM25 in DEFs,and challenged the cells at 24hours post transfection with NDV(5.43X10^5.64 copies/200?L).Then,we detecte d the NDV copies number at 24 and 48 hours post infection by qRT-PCR.Th e result indicated that the fusion protein DuRING-GoTRIM25 could significantl y inhibit the proliferation of NDV compare with GoTRIM25.4.The effect of TRIM25 in IFNs and ISGs mRNA expressionTo determine the effect of TRIM25 to IFNs and ISGs transcript,DEFs we re transfected with GoTRIM25 and DuRING-GoTRIM25,and then cells and vi rus were collected at 24 hours post infection for detection of IFNs and ISGs t ranscription level.Then,we detected IFNs and ISGs transcription level at 24 h ours post transfection by qRT-PCR.The result indicated that the fusion protein DuRING-GoTRIM25 could significantly up-regulate the IFNs and ISGs transcr ipt at 24 hours post transfection.