Effect Of Chicken CGAS On The Replication Of Avian Adenovirus Type 4 And The Establishment Of A DF-1 Cell Line For Stable Expression

Fowl adenovirus serotype 4(FAdV-4)is a genus Aviadenovirus with a genome size of43-45 kb and a capsidless double-stranded DNA virus.chcGAS(Chicken cyclic GMP-AMP synthase,chcGAS)is a natural immunomodulatory protein found in chickens that plays an important role in the fight against viral infections,especially DNA virus infections,by sensing the presence of DNA viruses and activating the innate immune response to participate in the development of the poultry industry.Whether FAdV-4,a DNA virus,can be recognized by chcGAS and perform antiviral functions remains to be resolved.Therefore,the aim of this study was to investigate whether chcGAS could activate the antiviral natural immunity and thus generate an immune response to induce a downstream cascade of immune responses against FAdV-4 infection,and to construct a DF-1 cell line stably expressing chcGAS,It provides a cellular model for the subsequent in-depth study of the mechanisms of fine regulation of the role of chcGAS in natural immunity.The main research contents and results are as follows:1.Cloning,expression,characterization and subcellular localization of chcGAS gene after FAdV-4 infectionIn this study,the chicken cGAS gene was successfully cloned and its biological properties were analysed using PCR.Bioinformatics software was used to predict the physicochemical properties,signal peptides and transmembrane structural domains of the protein encoded by the gene.Secondly,p ET-32a-chcGAS and p Cold-TF-chcGAS were expressed by E.coli expression system using the constructed prokaryotic expression vectors,and the proteins were identified by Western blot after expression.Finally,the constructed eukaryotic expression vectors pm Cherry-C1-chcGAS and p CAGGS-HA-chcGAS were used for cellular localization before and after FAdV-4 infection in LMH cells.The results showed that the chcGAS gene was 1317 bp in length and encoded 438 amino acids;bioinformatics analysis indicated that the protein was a stable hydrophilic protein with no signal peptide region or transmembrane structural domain,with 40 possible phosphorylation sites,and the secondary structure predicted that the protein contained four types of α-helix,β-turned angle,randomly coiled and extended chains.The analysis revealed that although the homology of cGAS was not high between species,the presence of Mab-21 in its structural domain made chicken cGAS evolutionarily conserved and the chicken cGAS was more closely related to duck and most distantly related to fish;the tissue distribution of expression showed that chcGAS was expressed in all tissues,with high expression in ileum,cecum and thymus,and lower expression in brain,trachea and kidney.The chcGAS protein was successfully expressed mainly in the supernatant;the changes in its localization before and after FAdV-4 infection were observed,with chcGAS mainly localized on the nuclear membrane when uninfected,but shifted from the nuclear membrane to the cytoplasm when infected.2.Study on the replication effect of chcGAS in FAdV-4 infectionTo investigate the effect of chcGAS on FAdV-4 replication,the eukaryotic expression vector p CMV-3×Flag-chcGAS was transfected into LMH cells and then infected with FAdV-4.The infected cell samples were collected and the expression levels of IFN-β,inflammatory factors and some antiviral protein genes were determined using RT-q PCR.The results showed that chcGAS could enhance IFN-β promoter activity and promote the expression of IL-6,IL-8,IFN-α,IRF7,TBK1,NF-κB,Mx-1,IFITM1,IFITM3.In addition,Overexpression of chcGAS into LMH cells,Cells were infected with FAdV-4 for 24 h of transfection.The copy number,protein expression level and FAdV-4 replication of FAdV-4 virus were detected by RT-q PCR,Westren blot and IFA techniques,respectively,and the analysis revealed that the expression of chcGAS showed some inhibitory effect on FAdV-4at different levels.3.Screening and identification of DF-1 cell lines stably expressing chcGASIn order to further investigate the role of chcGAS in natural immunity,DF-1 cell lines with stable expression of chcGAS were constructed by lentivirus packaging system.First,the recombinant lentivirus expression vector of chcGAS gene was constructed by homologous recombination technology.Then,the lentivirus expression plasmid and two auxiliary plasmids(p MD2.G and ps PAX2)were co-transfected into 293 T cells for lentivirus packaging to obtain chronic disease venom,and the DF-1 cells were infected.48 h after infection,purinomycin was used for continuous pressure screening until all cells showed green fluorescence,indicating the completion of cell line screening.The screened cell lines were identified by Westren blot,and the results showed that the stable expression of chcGAS was successfully constructed.In summary,In this study,we successfully cloned and expressed the chicken cGAS gene and analyzed the basic characteristics of the protein.Analysis of its localization revealed that when DNA virus FAdV-4 infection overexpressed chcGAS could be transferred from the nuclear membrane to the cytoplasm of the cell;To investigate the role of chcGAS in the induction of IFN-β,inflammatory factors,antiviral protein production and viral replication by FAdV-4 infection,and to clarify that chcGAS can regulate the antiviral natural immune response and thus have some inhibitory effect on FAdV-4 replication,It provides an important scientific basis for studying the regulatory mechanism of FAdV-4 in association with cGAS-STING signaling pathway;Finally,DF-1 cell lines stably expressing chcGAS were constructed in this study,providing valuable information for the in-depth study of the immune regulatory mechanism of chcGAS in avian species and its application in vaccine development.