A Preliminary Study On The Function Of DPV-US5 Gene Inhibiting Apoptosis Induced By H₂O₂

The virus infects the cells while the cells initiate programmed death to prevent the replication and spread of the virus.However,the virus also evolved a variety of survival strategies to neutralize apoptosis to coexist with the host cells,it is an effective mechanism for a virus to inhibit host nonspecific resistance.At present,the method of studying anti-apoptosis is mainly to construct the apoptotic model.The use of hydrogen peroxide,UV,MMC,Fas ligand,TNF-a-mediated apoptosis has been more mature.Treatment of duck embryo fibroblasts(DEF)for 12h with 400?mol/L hydrogen peroxide(H2O2).The nuclie become smaller,deform irregularly and apoptotic bodies appear with DAPI staining.The mRNA level of apoptotic factors caspase 3 and caspase 9 rise with qPCR detection rise(p*<0.05;n=3)·The activity of caspase 3/7 and caspase 9 protein enhance(p*<0.05;n=3).All of these results suggest H2O2 induce DEF apoptosis,and is associated with mitochondrial pathway.In this study,we first constructed the apoptotic model on duck embryo fibroblasts using hydrogen peroxide,which laid the foundation for the study of anti-apoptotic genes.Many genes of HSV-1 like US5 gene have been shown to be resistant to apoptosis.However,Duck plague virus(DPV),which also belongs to herpesvirus a subfamily,has no reports of anti-apoptotic function.Therefore,in this study,H2O2 was used to construct the apoptotic model on duck embryo fibroblasts for the first time,and on this basis,it was verified whether the duck disease virus gJ gene homologous to HSV-1 could inhibit apoptosis.The recombinant plasmid pCAGGS-US5 was constructed and identified by PCR and digested with restriction endonuclease.The recombinant plasmid pET-32a(+)-US5(M)was transformed into BL21 cells and expressed by IPTG.The purified gJ protein was used to immunize rabbits to obtain anti-DPV-gJ polyclonal antibody.The recombinant plasmid pCAGGS-US5 was transferred into DEF and treated with H2O2.The caspase3/7 protein activity reduce significantly with DPV-US5 expression compared with those who H2O2 treated alone.These suggest DPV-gJ can inhibit H2O2-induced apoptosis.