| Bovine viral diarrhea disease is caused by bovine viral diarrhea virus(BVDV), which is contagious disease. This disease distributed in the world widely and was serious harmful. The problem about persistent infection, immunosuppression molecular mechanism caused by BVDV, and the interaction between BVDV with the host cell still have not a systematic exposition. In vivo mi RNAs are key molecules that can regulate gene expression in post-transcription. Though mi RNAs have strong specificity and effictive characteristics, mi RNAs and their target sites has become a new bright spot in the design drug for the treatment of viral gene. We have detect the mi RNAs differences expression profile of MDBK infected with BVDV using solexa high-throughput sequencing. The expression profile showed that more than 20 mi RNAs expression was significantly increased. And after that we predicted three mi RNAs which targeted BVDV NADL genome by biology software. Therefore, we focused on exploring the molecular mechanism of micro RNAs affect the replication of BVDV NADL. Further to elucidate the molecular mechanisms of persistent infection of BVDV by which lay the theoretical foundation for the prevention and control of the new method.Objective: In this study, BVDV standard strain NADL servedas the research object. According to the difference mi RNAs in host cells that infected with BVDV NADL explore the molecular mechanisms of mi RNAs affect BVDV NADL replication.(1) to explore the mechanism of bta-mi R-33 a affecting BVDV NADL replication by targeting bim gene which was a part of BCL apoptosis pathway in MDBK cells;(2) to investigated the mechanisms of bta-mi R-302 c targeting BVDV NADL NS5 A gene which affect BVDV NADL replication;Methods:(1) According to the preliminary laboratory results of high-throughput sequencing compound 22 mi RNAs. And then transfected them into MDBK and infected BVDV in the same time. We screened the mi RNAs which can inhibited the replication of BVDV. We amplified the standard curves of BVDV NADL E0 Gene by q RT-PCR.(2) Biological information software predicted target genes of bta-mi R-33 a and verified the result with the dual luciferase reporter gene system. The protein and m RNA expression levels of bim was detected by Western blot,q RT-PCR.And the apoptosis of MDBK was detected by Flow cytometry after treated by bta-mi R-33 a. In the same time we detected BVDV NADL copy number changes by q RT-PCR. And then we constructed the green fluorescent overexpression lentivirus with bim gene.Using Western blot,q RT-PCR and Flow cytometry detecte the changes and the apoptosis of MDBK which overexpressing bim gene.And the changes of bim expression level was detected after treated by bta-mi R-33 a or BVDV.(3)(3) Prediction and identification of the target genes of mi RNAs in BVDV NADL genome with dual luciferase reporter gene system verification and Biological information software; Using q RT-PCR and titer detected the affection of bta-mi R-302 c, bta-mi R-205, bta-mi R-497 on BVDV NADL copy number and the TCID50. At different time intervals after treatment with BVDV NADL or treated by bta-mi R-302 c, the expression levels of JAK-STAT signaling pathway moleculars proteins and m RNA were detected using real-time quantitative PCR, Western blot.Results:(1) Bta-mi R-33 a,bta-mi R-27a-5,bta-mi R-21* can reduce the level of replication of BVDV NADL significantly. We constructed a standard curve of BVDV NADL E0 gene successfully.(2) Bta-mi R-33 a specific targeting bim gene 3 ’untranslated region(untranslated regions, UTR) of apoptosis pathway in MDBK; bta-mi R-33 a reduced apoptosis rate of MDBK and the copies of BVDV NADL by silencing bim gene expression; We constructed overexpression lentivirus of bim gene successfully; Bta-mi R-33 a can reduce the level of replication of BVDV NADL and the protein of bim in the MDBK that treated by the Bim-LV.(3) Bta-mi R-302 c target BVDV NADL of NS5 A 3 ’end specifically and directly; bta-mi R-302 c and bta-mi R-205 down-regulated m RNA levels of BVDV NADL E0 and bta-mi R-497 up-regulated m RNA levels of BVDV NADL E0; The expression level of factors in JAK-STAT pathway increased gradually as long time as the MDBK infected with BVDV NADL; bta-mi R-302 c can inhibit m RNA expression levels of factors in JAK-STAT pathway.Conclution: Bta-mi R-33 a down-regulated of bim gene which was part of apoptosis pathway protein Bcl-2-L11 protein family that result in the reduce of of apoptosis rate. With the affection of Bta-mi R-33 a, BVDV can not spread. Thereby Bta-mi R-33 a inhibited the replication of BVDV; bta-mi R-302 c targeting NS5 A 3 ’end of the BVDV NADL, regulated JAK-STAT pathway indirectly, which promote cells expressing anti-viral mechanism and inhibit BVDV NADL replication. |
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