Research Of The Apoptosis For Bcl-2 Like Protein ORFV125 Inhibition Of Host Cell Apoptosis

Contagious ecthyma,as a zoonotic infectious disease caused by Orf virus(ORFV)in sheep and goats,Western China is the main area of disease.After the infection,the mouth,nose and breasts of diseased animals have pustules and scabs.Shemorrhoids has become an important pathogen in the prevention and control of livestock diseases in china,and has received extensive attention from domestic and foreign scholars.The ORFV encode Bcl-2like proteins ORFV125.Previous study shows that ORFV125 and Bcl-2 protein family have highly similar 3D structure and domain,and Bcl-2 have anti-apoptotic function.Therefore,the aim of this study was to explore whether ORFV125 have anti-apoptotic function.In the study,Luoyang strain was downloaded according to the NCBI website,and the entire ORFV125 gene was ligated into the pCAGGS vector to construct a mutant plasmid containing the recombinant plasmid pCAGGS-ORFV125 and its domain were synthesized in the company.The ORFV125 and its mutants were then transfected into PK-15 cells and cultured for 24 h.The cells were treated with JC-1 dye and observed under a fluorescence microscope.The results showed that the cells had green fluorescence,indicating changes in mitochondrial membrane potential.The protein interacted with ORFV125 was initially screened by yeast two-hybrid,and the interaction between ORFV125 and the inhibitory apoptotic protein Birc5 was verified by(Co-Ip)method.Co-immunoprecipitation results showed that there was a blot of the interaction between ORFV125 and Birc5 on the NC membrane.The ORFV125 and its mutant plasmids were transfected into 293 T cells and cultured in groups of 24 h and 72 h respectively,and normal cells were set as control group.The cells of each group were treated with trypsin digestion solution,stained with Annexin V-FITC and PI,and the samples were detected by flow cytometry.The results showed that the percent of early apoptosis was 22.65% and the percent of late apoptosis was 5.51% in control group at the 24 h transfection time;the percent of early apoptosis was less than 5% in the experimental group and the percent of late apoptosis was less than 6%.At 72 h transfection time,the percent of early apoptosis was 32.06% and the percent of late apoptosis was 3.22% in control group;the percent of early apoptosis was 24.62%-48.29% in the experimental group and the percent of late apoptosis was 2.12%-3.68%.The above studies preliminarily demonstrated that ORFV125 interacts with the inhibitory apoptotic protein,Birc5,and verified that ORFV125 can inhibit apoptosis.It was further proved that ORFV125 and its mutants had the effect of inhibiting apoptosis after transfected into cells,and the apoptosis ratio was time-dependent.