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The Analysis Of BVDV NS3 Protein Antigenic Site And Its Effect On The Regulation Of IRES-Mediated Translation Mechanism

Posted on:2019-05-01Degree:MasterType:Thesis
Country:ChinaCandidate:X K ZhouFull Text:PDF
GTID:2370330548464054Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Non-structural protein NS3 of Bovine viral diarrhea virus(BVDV)plays an important role in virus replication,protein synthesis,and apoptosis of host cells.The translation of the BVDV open reading frame depends on the internal ribosomal entry site(IRES)in the upstream non-translated region(5'UTR).BVDV NS3 may participate in and influence the replication cycle of BVDV virus by regulating the translation of the virus mediated by the internal ribosome entry site(IRES).During the proliferation of BVDV after infection,NS3 protein can be recognized by CD4+T cells in the host,thus inducing antibodies against NS3 protein.Through DNA-star analysis,the B cell antigen epitope and T cell antigen epitope of NS3 protein can be calculated,on the basis of NCBI database the nucleotide sequences of the NS3 protein,we construct the NS3 prokaryotic expression vector named pET-28a-his-NS3,then,It proved that the NS3 had antigenicity after react with infected BVDV positive serum.This lay foundation for the establishment of rapid immunological detection methods and vaccine development against BVDV.At the same time,we construct eukaryotic expression vector pEGFPC2-NS3 and double report carrier pcDNA3.1-CAT-IRES-GFP BVDV IRES.We found that NS3 can down regulate IRES mediated GFP protein expression level with dose-dependent manner.In order to further determine the interact area of NS3 and IRES,the luciferase report carrier IRES pGL6-D and segmented luciferase report expression vector,pGL6-A?pGL6-B?and pGL6-C were built,then,according to dual luciferase report experiments and Western-blot method.Each function structure domain of IRES mediated translation level have the same function,and with the increment of NS3 expression,the IRES and IRES segmented structure domain mediated luciferase activity gradually reduced.It seemed that NS3 protein have negative regulation effect on IRES mediated translation mechanism in dose-dependent manner.In conclusion,this study illustrates that the each structure of IRES for is indispensable,so,it is of great significance for the structural integrity of IRES in IRES mediated translation mechanism.
Keywords/Search Tags:Bovine viral diarrhea virus, Non-structural protein NS3, Internal ribosomal entry site, Reporter gene, Translation regulation
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