Study On Non-structural Protein NS1 Of Canine Parvovirus Inducing Apoptosis In Host Cells

Canine parvovirus disease is an acute infectious disease which caused by canine parvovirus (CPV). Acute hemorrhagic gastroenteritis and acute myocarditis with high morbidity and mortality is the most common manifestation of the disease, which is harmful to dogs, especially to puppies. Therefore, it is an very important infectious disease. However, the molecular mechanism of canine parvovirus disease is not entirely clear.It has been proved that CPV-infection can induce apoptosis in intestinal epithelial cells from CPV-infected animals, and thereby cause secondary infection. To investigate the effects of canine parvovirus non-structural protein-1 (NS1) on the cell apoptosis induced by CPV and preliminarily explore the mechanism of CPV-induced apoptosis, the expression of NS1 protein is needed. We selected eukaryotic expression system to obtain NS1 protein with biological activity.First,the NS1 gene was amplified by PCR from CPV genomic DNA and subcloned into pcDNA3.1A vector to generate NS1 eukaryotic expression vector pcDNA-NS1/His and pcDNA-NS1. To verify whether pcDNA-NS1/His vector can mediate NS1 expression in eukaryotic cells, the human embryo kideny (HEK) 293FT cells were used to transiently express the recombinant NS1 protein. The effects of NS1 on CPV-induced apoptosis were investigated by infecting the F81 host cells with CPV and transfecting the cells with NS1 vector. The apoptosis of the cells was detected by AnnexinV/PI double staining for phosphatidylserine externalization on membrane and by luminescence method for caspase-3/7 activities.The results show that the sequence of NS1 gene amplified was consistent with the GenBank. The NS1 expression vector was shown to be correct and could mediate NS1 expression in eukaryotic cells. The phosphatidylserine (PS) on outside of membrane was detected and the caspase-3/7 activities were increased in both CPV-infected cells and NS1-transfected cells. These results indicate that both CPV and NS1 protein can induce the apoptosis of the cells. In summary, canine parvovirus NS1 protein can induce apoptosis, which laids the foundation for further investigation of the molecular mechanism of canine parvovirus disease.