Study On The The Molecular Mechanisms Of The Copy Of BVDV Intracellular Affected By MiR-193a In Cow

The molecular mechanisms of BVDV induced persisting infection has not been studied well, the most important thing is that the interaction mechanism of BVDV and the host cell has not been clearly explained. Numerous studies showed that when the virus invade cells, cells will initiate apoptosis, or apoptosis is induced, which led to the death of infected cells and virus were removed. Previous experiments of our laboratory about using solexa high-throughput sequencing detect the expression profiles after BVDV infecting the MDBK showed: The expression of mi R-193 a has been significantly improved, and the bioinformatics software showed the target gene of mi R-193 a on apoptosis is the Bax. Therefore, our study treated the mi R-193 a as the object, to explore the molecular mechanisms of apoptosis and the replication of BVDV. Lay the theoretical foundation for the prevention of persistent infection of BVDV and new ways of BVDV exposition.[Objective] Explore the impact of miR-193 a target protein on apoptotic pathways and the molecular mechanisms of BVDV replication.[Method](1) Based on our previous high-throughput sequencing results,We chose the mi R-193 a which significant upregulated in the previous experiments;(2)Clon the cloning of mi R-193 a and its inhibitor the mi R-193a-IN. Then build the pll3.7-mi R-193 a and pll3.7-mi R-193a-IN recombinant vectors, and they were transfected into MDBK cells. The effection of overexpression and inhibition mi R-193 a were detected by q RT-PCR.(3)The mimics and inhibitor of mi R-193 a were transfected into MDBK cells, then detected the copy number of BVDV by q RT-PCR.(4)Predicted the target genes of mi R-193 a by bioinformatics software, then build the pmir GLO-Bax 3’UTR recombinant vectors, and transfect the pll3.7- mi R-193 a with pmir GLO-Bax 3’UTR into MDBK cells, then detect the fluorescent activity.(5) After overexpression and inhibition of mi R-193 a, detect Bax transcription level by RT-PCR, and detect the expression level of Bax protein by western blotting and the apoptosis rate was detected using flow cytometry;(6) The primers were designed using the software in pre-mi R-193 a upstream, amplified them, then build the p GL3-Basic-gene recombinant vectors.Transfect it into 293 T cells, then detect the fluorescent activity.(7) According to the promoter sequence design methylation-specific primers, make the methylated DNA as the template, amplificate it, connect it with T vector, send them to company start methylation sequencing.(8)Design the sh RNA of interfere methylation sites, construct recombinant vectors of sh RNA- lentiviral, and they were transfected into MDBK cells. Extract the whole genome, make the methylated DNA as the template, amplificate it, connect it with T vector, send them to company startmethylation sequencing.(9) using fluorescence quantitative micro RNA detection kit detected the expression levels of mi R-193 a after sh RNA- lentivirus infected the MDBK cells.[Result](1) Overexpression of mi R-193 a upregulated the expression of mi R-193 a, mi R-193-IN inhibited it’s expression.(2)The mimics of mi R-193 a were transfected into MDBK cells, decreased the copy number of BVDV.(3) Confrim that Bax was the target gene of mi R-193 a.(4) The cells overexpress mi R-193 a increase apoptosis rate,and Bax is downregulated.(5)Successfully constructed the p GL3-Basic-gene luciferase recombinant vector,and verified the mi R-193a-P4 is the promoter of mi R-193 a.(6)Admix the sh RNA lentiviral recombinant vectors, analyze the results of methylation detection by software. The result shows: the degree of mi R-193 a methylation of MDBK cells that BVDV is lower than normal MDBK cells about 19%. It proved that infection of BVDV leads the demethylation of mi R-193 a.(7)The degree of mi R-193 a methylation of MDBK cells that infect sh RNA- lentivirus shows:pll3.7-193a-promoter sh RNA-2 is the lowest, It promotes the expression of mi R-193 a, this section area most likely to be the promoter of mi R-193 a.(8) Micro RNA quantitative fluorescence showed that the highest expression level of mi R-193 a in the MDBK cells was the pll3.7-193a-promoter sh RNA-2 led.[Conclusion](1) The possible mechanism of mi R-193 a promotes apoptosis and inhibits BVDV replication is that mi R-193 a induces apoptosis by Bax, so the apoptotic bodies can be formated, then phagocyte swallows up the apoptotic bodies, lysosome will fuses and degradates of the virus,so the mi R-193 a inhibits the replication of BVDV.(2)Determining the mi R-193a-P4 is the possible promoter of the mi R-193 a. After the infection of BVDV may lead to the demethylation of mi R-193 a promoter region, then the expression of mi R-193 a may be up-regulated; pll3.7-193a-promoter sh RNA-2 can significantly reduce the degree of methylation, which can increase the expression of mi R-193 a, make the possible promoter region of mi R-193 a more more accurate, then we can lay the theoretical foundation for the effective regulate the expression of mi R-193 a.