The Study Of MiR-2459 Affecting BVDV Replication Through Regulating Zinc Finger Antiviral Protein ZAP Expression

At present,the molecular mechanism of persistent infection caused by BVDV has not been studied clearly.The interaction between BVDV and host cells has not been systematically described.Therefore,in our previous study,using Solexa high-throughput sequencing to detect the miRNA expression profiles after target cells MDBK rinfected by BVDV.The results indicated that the expression of miR-2459 increased significantly in cells which infected by BVDV.Then we predicted that miR-2459 targeted to zinc finger antiviral protein gene ZAP by bioinformatics prediction software analysis.Therefore,In this study takes miR-2459 as the study object,to explore how miR-2459 affects BVDV replication? What role does ZAP play in it? and to lay a theoretical foundation for the elaboration of BVDV's persistent infection and BVDV prevention and control.Objective:In order to explore the molecular mechanism of miR-2459 promoting BVDV replication.The results of high throughput sequencing of BVDV cells infected by BVDV were analyzed,and selected miR-2459 whose expression level was up-regulated to further study.Methods:?After BVDV infected MDBK cells in short term,using the method of Stem-loop RT-PCR to identify the expression of miR-2459 in cells;After transfected miR-2459 mimics and inhibitors,To detected the level of BVDV replication in RT-PCR and Reed-Münch.?The target genes of miR-2459 were predicted by bioinformatics software;It identified that miR-2459 directly targeted to ZAP 3 'UTR region by the dual luciferase reporter assay;Detected the expression of ZAP after transfected miR-2459 mimics and inhibitors in RT-PCR;Detected the expression of ZAP after MDBK cells were infected by BVDV NADL in RT-PCR.? Constructed overexpression ZAP vector p LEX-EGFP-ZAP;Coated lentivirus by the calcium phosphate transfection way in HEK-293 cells;After lentiviral infecting MDBK cells and screening the cells use puromycin,and finally obtained stable MDBK cell lines that over expressing ZAP;After BVDV infected MDBK cells of overexpress ZAP,detecting the effect of miR-2459 on BVDV replication level in RT-PCR and Reed-Münch.Results:?Identificated the MDBK cells infected by BVDV in a short time,the expression level of miR-2459 cells were increased in Stem-loop RT-PCR;Infected BVDV after transfected miR-2459 mimics and inhibitors in MDBK cells,and miR-2459 mimics promoted BVDV replication.?The target gene of miR-2459 is antiviral related zinc finger protein gene in Bioinformatics software;The luciferase reporter assays identified that miR-2459 directly targeted to ZAP 3 'UTR region;After BVDV NADL infected targeted cells,the expression level of ZAP was decreasing;After transfected miR-2459 mimics and inhibitors,the expression of ZAP was inhibited by miR-2459.?Constructed ZAP overexpression vector p LEX-EGFP-ZAP successfully;Coated lentivirus and obtained MDBK cell line of stable overexpressing ZAP;MDBK cell that overexpress ZAP has a significantly inhibitory effect on BVDV replication in RT-PCR and Reed-Münch.Conclusion:miR-2459 directly targets to the 3 'UTR region of zinc finger protein ZAP,and affects the expression level of ZAP,thereby indirectly inhibiting the antiviral effect of cells.Play an important positive role in BVDV replication.