Mink Aleutian Virus VP2 Protein Induces Apoptosis Of CrFK Cells And Screening Of SiRNA Targeting VP2 Gene

Aleutian mink disease(AMD)is a chronic and infectious disease caused by Aleutian mink disease virus(AMDV).It is one of the main diseases that cause economic losses in the global mink farming industry.AMDV can encode three nonstructural proteins(NS1-NS3)and two structural proteins(VP1 and VP2),of which VP2 protein is the main immunogenic protein and plays an important role in virus invasion,pathogenicity and host selection.Therefore,this study aims to explore the effect of VP2 protein on cell apoptosis and screen siRNA targeting VP2 gene.In order to construct the eukaryotic expression vector of the mink Aleutian virus VP2 gene,the AMDV-G strain VP2 gene was linked to the vector pEGFP-C3,and then transfected into CrFK cells,the m RNA and protein levels were detected;the laser confocal technique was used to detect the VP2 protein Distribution in CrFK cells.The results showed that the AMDV VP2 gene eukaryotic expression vector pEGFP-C3 VP2 was constructed and transfected into CrFK cells,and the fluorescence intensity and VP2 protein expression increased with time;laser confocal microscopy showed that pEGFPC3 VP2 The green fluorescence of the transfection group was distributed in both the cytoplasm and the nucleus,but the distribution in the cytoplasm increased significantly,indicating that AMDV VP2 protein was distributed in the cytoplasm and nucleus of CrFK cells.To investigate whether the expression of mink Aleutian virus VP2 gene can cause CrFK cell apoptosis,the constructed pEGFP-C3 VP2 recombinant plasmid was transfected into CrFK cells,and studied by fluorescence quantitative PCR,Caspase cell viability experiment,TUNEL staining,Hochest33258 staining and flow cytometry.The results showed that after transfection of CrFK cells with pEGFP-C3 VP2 recombinant plasmid,the expression of apoptosis executive factor Caspase-3 increased with time,which was the same results of the cell viability experiment of Caspase-3.The results of TUNEL staining and Hochest33258 staining both showed that the pEGFP-C3 VP2 recombinant plasmid transfected CrFK cells for 48 h can cause CrFK cell apoptosis.The results of flow cytometry showed that the apoptosis rate of the pEGFP-C3 VP2 recombinant plasmid transfected group was significantly higher than that of the empty vector group pEGFP-C3 and the blank cell control group.The above results indicated that the expression of mink Aleutian virus VP2 gene in CrFK cells can cause cell apoptosis.In order to screen for siRNA targeting AMDV-G strain VP2 gene and study whether it can reduce cell apoptosis caused by virus infection,three interfering RNAs targeting AMDV-G strain VP2 gene were designed and synthesized,fluorescence quantitative PCR technology,indirect Immunofluorescence test,TCID50 determination,CCK-8 method and flow cytometry were used for research.Fluorescence quantitative PCR test results showed that the transfection dose of siRNA1 was 5 μL,and the interference had a significant inhibitory effect on virus proliferation at 48 h;indirect immunofluorescence test,TCID50 determination,and CCK-8 method results showed that siRNA1 and siRNA2 can significantly inhibit AMDV proliferation in CrFK cells;flow cytometry results show that both siRNA1 and siRNA2 can inhibit cell apoptosis caused by virus infection,and siRNA1 had a more obvious inhibitory effect.In summary,this study provides new data for the study of the pathogenic mechanism of AMDV and lays the foundation for further research on the prevention and treatment of Aleutian disease in mink.