| 【Objective】Rabbit hemorrhagic disease (RHD) causes high mortality in both domesticand wild adult animals, RHDV is a serious viral disease in the rabbit industry.The basicresearch of rabbit hemorrhagic disease virus (RHDV) could benefit the control of disease，unfortunately，the study of rabbit hemorrhagic disease virus (RHDV) has long been hinderedby the absence of an in vitro culture system.Recent researches revealed that it，theCaliciviridae have some specialties in its biological characteristics，it is possible to propose amodel of novel mechanism of calicivirus VPg-dependent proteinsynthesis.In this study,wemade a Preliminary study in viruses translation mechanism by reverse genetics technology.【Method】(1) The VPg gene from pBL-RHDV plasmid（JX/97） was cloned intoexpression vector pET-30a,and the recombinant plasmid pET-30a/VPg was transfected intoE.coil BL21cells,then induced by IPTG, RHDV VPg was purified by affinity chromatographyon NiCam-agarose, followed by vaccinated into rabbit to produce polyclonal antibodies,theanti-serum was collected and detected by western blot.（2）Based on the full-length cDNAclone of RHDV，a series of DNA-based reporter replicons were constructed in which thefirefly luciferase (Fluc) gene was fused in-frame with the open reading frame of the replicon.the replicon is under the control of a minimal cytomegalovirus (CMV) immediate-earlypromoter，The replication of the replicon in transfected RK13cells was detected by IFA,RT-PCR and replication dynamics of RHDV replicon；On the basis of the replicon,5’non-coding regions (5’NCR) genome-linked protein (VPg) and3D were deleted, and theeffect on the expression of replicon was analyzed by expression level of Fluc.To explore therelation between VPg/5’NCR and viral translation，On the basis of replication deficient mutantreplicon（Δ3D），the impact of5’NCR and VPg deletion on viral translation efficiency wasanalyzed by dual-luciferase reporter assay system， we also performed qRT-PCR annalysis ofRNA isolated from samples，In summary，all of this studies attempted to identify factorsassociated with RHDV translation.【Result】(1)We constructed the Prokaryotes expression vector of pET-30a/VPg, the protein was high levels soluble expression in E.coil BL21cells,the western blot showedspecificity of the antibody.(2) We constructed a plasmid containing an Fluc gene fusedin-frame with the viral ORF in the position where the structural genes were deleted，theresults confirmed the ability of RHDV replicon automatically replicate in RK13cells. Timecourse analysis showed that the intensity of the luciferase signal reached a maximum value at24h post-transfection. In this study，we have utilized the system for analyzing the potentialeffect of5’NCR on viral translation. Our results showed that5’NCR played an important rolein viral transcription and translation. Furthermore，in the replication deficient mutantreplicon，either deletion of VPg or5’NCR could severely impaired the level of flucproduction，and the reduce on the level of luciferase activity does not rely on the mRNAconcentration, the results prove that the VPg and5’NCR are essential for RHDV translation.【Conclution】This research provides good material base for further studying thebiological characteristics of VPg；(2) The results of this study demonstrated that the reporterreplicon replicates efficiently in mammalian cells and the expression level of the repliconreporter gene correlates to the level of genome replication and translation,the replicon hasconsiderably facilitated genetics analysis of RHDV replication and translation (3) based onthe replicon,the study strongly suggest that the luciferase activity was reduced in the absenceof5’NCR, VPg and3D,and VPg and5’NCR are involved in viral transcription andtranslation. |
PDF Full Text Request