Complete Genome Sequencing And Construction Of Recombinant Swinepox Virus Expressing E2 And E0 Protein Of Classical Swine Fever Virus

Classical swine fever was a highly contagious diseases caused by classicalswine fever virus(CSFV),which was listed in A diseases by Office International des épizooties(OIE).CSFV was single postive RNA virus which belongs to flaviviridae and pestivirus.Swine fever virus envelope protein E2 has good immunogenicity,which can induce to produce neutralizing antibody and provide immune protection sufferd from virulent strain.Envelope protein E0 can also induce body to produce neutralizing antibody and provide some immune protection.This study investigated theepidemiology of CSFV E2 gene molecular of Jiangxi province,sequencing and analyzed of the complete genome,and constructed the recombinant swinepox virus that expressing E2 and E0 protein of CSFV,which offers the basis to control classical swine fever.1.Investigation the CSFV E2 gene molecular epidemiology of Jiangxi provinceThis study collected 26 positive swine fever samples in Jiangxi province in 2014 and 2015.Used RT-PCR method,we amplified and sequenced E2 gene.Compared to the standard reference strains.Result shows that all of the samples belong to 2.1,in addition 22 samples belong to 2.1d subgenotype and 4 samples belong to 2.1b.2.Sequencing and analysis of the whole genomeThis study isolated CSFV strains from pathological sample,and we designedeight nested PCR primers that cover the genome and amplified the sequence of nine strains and sended to sequence.Compared to the reference sequence of each subgenetype.we used complete genome,5'NTR,four structure protein,eight nonstructure protein and 3'NTR to establish phylogenetic tree,and E2 gene re garded as the reference.Result shows that complete genome and Npro,NS4 B,E2 and NS3 gene could be the basis of genetyping.E0,E1,NS2,NS5 A,NS5 may be a new way to genetyping.5'UTR,C,3'UTR,P7 and NS4 A can not correctly use for genetyping.Used E2 gene as the reference,We genetyped the nine strains isolated in this study.Result shows that seven strains belong to 2.1d and two belong to 2.1b.3.Construction of recombinant swinepox virus that express ERFPThis study aimed to construct the recombinant swinepox that express enhance red fluorescent protein(ERFP).Firstly we amplified left and right arms that recombinant swinepox virus needed,and then amplified with ERFP with swinepox virus late promoter P11 fragment.The three fragment was fusion to a fragment through fusion PCR method,than this fragment was clone in pUC19 though restriction enzyme digestion,than we transfect it to PK15 pre-infected swinepox virus.Result shows that sequencing and restriction enzyme digestion result wre identify with the prospection and ERFP were effectively expressed in swinepox virus,which indicated that the transfer vector was successfully constructed and named pSW-ER11.This study lay foundation to purify recombinant swinepox virus used ERFP as label.4.Construction of recombinant swinepox virus that expressing CSFV E2 and E0This study aimed to construct the swinepox transfer vector that express CSFV E2 and E0 protein.Firstly we cloned E2 and E0 gene from CSFV genome,all gene were regulated by poxvirus later promoter.Secondly,we cloned into the destination framents into linearized pSW-ER11 by in-Fusion clone step.Result shows that digestion and sequencing results are identify with the prospection,which indicates the respective swinepox transfer vectors have been successfully constructed.The recombinant virus provides basis to explore the protective immunity of classical swine fever virus E2 and E0 protein expressed by swinepox virus.5.Reform the plasmid of pcDNA3.1+This study aimed to reconstruct the pcDNA3.1+,Firstly we cut down the CMV promoter fragment which contain T7 RNA polymerase promoter sequence,later we cloned the CMV promoter fragment which not contain T7 RNA polymerase promoter sequence to the vector,by this way we deleted T7 RNA polymerase promoter sequence of the vector in order to prevent transcription the sequence of the vector into the CSFV RNA.Result shows that T7 RNA polymerase promoter of the pcDNA3.1+ was successfully deleted.It lay foundation to construct infectious clone of CSFV.