Construction Of Recombinant Encephalomyocarditis Virus Expressing COE Protein Of PEDV

Porcine epidemic diarrhea?PED?is a highly contagious disease which causes by porcine epidemic diarrhea virus?PEDV?with diarrhea,vomiting and dehydration as the main symptoms.PED broke out around the world,and caused huge economic losses to the global pig industry.PEDV S protein is a kind of fibrin protein,which is located on the surface of virus particles.It plays an important role in the process of virus propagation,genome diversity and genetic variation.It is the target protein for clinical diagnosis and vaccine development.The PEDV S protein is divided into S1?1-789 aa?and S2?790-1383aa?regions.An important epitope region?499-638 aa?of PEDV is designated as COE protein in S1region,and COE protein is used as a target protein in the genetically engineering vaccine.With the development of reverse genetic system,it makes possible to use a single positive stranded RNA virus as a live vector for genetically engineering vaccines.Encephalomyocarditis virus?EMCV?belongs to the Cardiovirus genus of the Picornaviridae family,which is an important zoonotic pathogen.Piglets mainly cause acute myocarditis piglets,and the mortality rate of up to 100%;Sows infecte EMCV mainly cause reproductive disorders.In addition,EMCV can transmiss in interspecies,people pay more attention to the influence of public health.The recombinant virus expressing PEDV COE protein was constructed by using EMCV as the vector,so as to achieve the goal of preventing the two diseases.In this study,EMCV full-length cDNA was amplified by RT-PCR using EMCV HB10 strain as template and cloned into pBluescript SK II vector to construct recombinant vector pBSK-rEMCV.The pBSK-rEMCV transcripted into genomic RNA in vitro and the RNA transinfected into BHK21 cells for viral rescue.By RT-PCR,Western Blot and sequencing identification,which proved that rescue of the rEMCV is successful.In addition,the rescued rEMCV maintained the parental toxicity of HB10 on BHK21 cells.The construction of EMCV full-length infectious cDNA clones provides a powerful tool for the research of EMCV,such as molecular pathogenesis,vaccine construction,gene function and other viral molecular biology.On the basis of EMCV full-length infectious cDNA clone,the recombinant EMCV expressing PEDV COE protein was further constructed.The COE gene was inserted into the EMCV genome by fusion PCR to construct pBSK-rEMCV-COE recombinant vector.The pBSK-rEMCV-COE transcripted to the EMCV genomic RNA with COE gene in vitro and the RNA transfected into BHK21 cells to rescue the recombinant virus rEMCV-COE.Indectification of the rENCV-COE by RT-PCR,Western Blot,indirect immunofluorescence and sequencing analysis,which proved that rescue of the rEMCV-COE is successful.The r EMCV-COE showed good adaptability and stability on BHK21 cells.The rEMCV-COE challenged mice by intramuscular injection at a dose of 10¹ TCID₅₀/mouse.The antibodies against EMCV and PEDV were detected at 3d,7d,14d,21d and 28d after challenged rEMCV-COE by ELISA kit.The results showed that mice produced antibodies of EMCV and PEDV,and the level of antibodies reached maximum at 14d²1d post challenged days.The recombinant pBSK-rEMCV-COE vector provides a reference for the development of EMCV and PEDV live vaccine in the future.