| Bovine viral diarrhea(BVD)is an infectious disease caused by Bovine viral diarrhea virus(BVDV).It is widely spread,not only through direct contact transmission,but also through various excreta,contaminated materials,etc.The infection of BVDV not only causes huge economic losses to the aquaculture industry,but also hinders the development of the serum related biological product industry.Many countries have introduced control measures to purify BVDV,but its prevalence remains high in old homes.Apoptosis plays an important role in limiting the infection of multiple pathogens.Therefore,this study found that BVDV infection can induce apoptosis of MDBK cells.proteomics technology was used to screen TOP2 A,a differential protein related to apoptosis,and study its effect on cell apoptosis.1.Analysis of BVDV infection regulating MDBK cell apoptosis.1 MOI NADL was used to infect MDBK cells,and the cell samples were collected at 6 h,12 h,24 h,36 h and 48 h,respectively.The results showed that the apoptotic execution factor Caspase-3 expression was upregulated and the Bax/Bcl-2ratio was upregulated by RT-q PCR and Caspase-3 activity assay,respectively;further application of FITC and PI staining and flow cytometry detection revealed that after NADL infection,the number of apoptotic cells increased with time,and early apoptosis occurred at 12 hours.2.Proteomic analysis of BVDV infected MDBK cells.1 MOI NADL strain was used to infect MDBK cells,and cell samples were collected at 6 h,12 h,24 h,36 h and 48 h after infection for proteomic sequencing,and a negative control group was set up.The sequencing revealed that 323 differential proteins were significantly up-regulated and 203 proteins were significantly down-regulated in the NADL 6 h/NC comparison group;406 differential proteins were significantly up-regulated and 197 proteins were significantly down-regulated in the NADL 12 h/NC comparison group;255 differential proteins were significantly up-regulated and 208 proteins were significantly down-regulated in the NADL 24h/NC comparison group;568 differential proteins were significantly up-regulated and208 proteins were significantly down-regulated in the NADL 36 h/NC comparison group.There were 568 differential proteins significantly up-regulated and 201 proteins significantly down-regulated in the NADL 36 h/NC comparison group;663proteins significantly up-regulated and 341 proteins significantly down-regulated in the NADL 48 h/NC comparison group.GO enrichment analysis and KEGG enrichment analysis of differential proteins revealed that differential proteins at different infection times were significantly enriched in biological processes,cellular components and molecular functions,in addition,apoptosis-related terms were enriched in five time points;pathways were concentrated in biosynthesis,metabolism,immunity and disease.The results showed that there were 58 up-regulated differential proteins and 29 down-regulated differential proteins in NADL 6 h/NC,54up-regulated differential proteins and 16 down-regulated differential proteins in NADL 12 h/NC,43 up-regulated differential proteins and 21 down-regulated differential proteins in NADL 24 h/NC,82 up-regulated differential proteins and 22down-regulated differential proteins in NADL 36 h/NC.NADL 48 h/NC had 100up-regulated differential proteins and 41 down-regulated differential proteins,respectively.The three differential proteins BAG3,OPTN and TOP2 A were selected and validated by Western Blot assay,and the results were consistent with the sequencing results.3.Effect of TOP2 A protein on apoptosis.Based on the results of proteomic sequencing,TOP2 A protein was found to play an important role in the biological process of apoptosis.The optimal action concentration of Pluri SIn #2 was determined by CCK-8,RT-q PCR and Western Blot assay at 30 μM.Mitochondrial membrane potential assay revealed that TOP2 A inhibition caused mitochondrial damage;TUNEL,Hoechst staining and flow cytometry assay revealed that inhibition of TOP2 A protein expression resulted in a significant increase in the number of apoptotic cells increased.The results indicated that inhibition of TOP2 A protein could cause mitochondrial damage and thus induce apoptosis.In summary,this study applied BVDV NADL infection to MDBK cells to verify the occurrence of apoptosis;applied proteomics technology to screen for the apoptosis-related differential protein TOP2A;further verified that TOP2 A inhibition contributed to the induction of apoptosis in MDBK by NADL. |
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