The Deletion Of Swinepox Viruses TK Gene&Construction Of Recombinant Swinepox Virus Expressing VP60 Protein Of RHDV

Swinepox virus(SWPV)is the sole member of the genus Suipoxvirus,belonging to the family Poxviridae.SWPV infects only swine.Natural SPV infections are typically mild and occasionally accompanied by localized skin lesions that heal naturally.SPV is well suited for the development of recombinant vaccines,because of its biological and clinical safety,large packaging capacity for recombinant DNA,and its ability to induce solid protective immunity.Rabbit Hemorrhagic Disease(RHD)cause by Rabbit Hemorrhagic Disease Virus(RHDV),is a highly contagious rabbit fatal disease.The current domestic application of RHD vaccine is tissue inactivated vaccine,but genetically engineered vaccine is the future direction.B ased on swine pox virus can non-replicative multiply in rabbits,VP60 protein is the major structural protein of RHDV and closely related to the immunogenicity and pathogenicity of RHDV.in order to investigate the feasibility of Swinepox virus as a vector construct recombinant live vector vaccine RHD.In this study,Jiangxi swine pox virus isolates strain JX-20G as parental virus,TK gene as an insertion site for a foreign gene,EGFP/LacZ as selection marker,by homologous recombination technology,established a recombinant Swinepox virus technology platform.On this basis,constructed recombinant Swinepox virus that can express RHDV VP60 protein,and determinat the biological properties of recombinant VP60 protein.1.Cloning and expression the SWPV VP60 geneAccording to RHDV VP60 gene,designed and synthesized a pair of primers,after RT-PCR amplification,T carrier connections and sequence analysis,The results show the homology of nucleic acid sequences between isolates and domestic and foreign reference strain was 91.8%-99.8%;The deduced amino acid homology was 97.8%-99.8%.Used prokaryotic expression vector pET-3 2a(+),constructed recombinant expression vector pET-32a(+)-VP60,after Ni-affinity chromatography purified, SDS-PAGE showed the recombinant protein His-VP60 size is about 80 ku.Coated microtiter plates with recombinant proteins,detected by RHDV positive and negative serum,The results showed that the recombinant protein had good reactogenicity,and can used to RHDV antibody ELISA detection.2.Green fluorescent protein gene as a selection marker construct TK deletion swinepox virusJiangxi Swinepox virus isolates strain JX-20G as parental virus,TK gene as an insertion site for a foreign gene,EGFP as selection marker,Vaccinai virus(VV)P11 promoter as exogenous gene promoter,enestablished a transfer vector pSWE11,and verified by PCR and restriction enzyme digestion.By homologous recombination technology,obtained a TK gene deletion recombinant virus rSWPV-Ell.Recombinant viruses were identified by PCR,fluorescence observation and analysis of genetic stability.Fluorescence observation showed that the recombinant virus produced fluorescence,and expressed the green fluorescent protein,indicated that the P11 may action as a promoter for recombinant poxvirus swine promoter exogenous gene.The recombinant virus 10 generations,produced fluorescence was no change,indicated that deletion of the TK gene can be Swinepox virus JX-20G exogenous fragment insertion sites.3.LacZ gene as a selection marker construct TK deletion swinepox virusLacZ gene as selection marker,P11 promoter and P28 promoter as exogenous gene promoter,enestablished two transfer vector pSWZ11 and pSWZ28,and verified by PCR and restriction enzyme digestion.By homologous recombination technology,obtained two recombinant virus rSWPV-Z11 and rSWPV-Z28.Recombinant viruses were identified by PCR,blue plaques observation and analysis of genetic stability.The results showed that in the course of virus purification and screening,both recombinant viruses produced blue plaques.After 10 generations of recombinant virus,can produce blue plaques,indicating two recombinant viruses have good genetic stability.4.Construction of recombinant swinepox virus expressing RHDV VP60 proteinIn order to explore the potential of swine pox virus as a RHD vaccine vector,constructed two transfer vector pSWE11-7.5V and pSWE11-28V,By homologous recombination technology,obtained two recombinant virus rSWPV-E11-7.5V and rSWPV-E11-28V.Recombinant viruses were identified by PCR,genetic stability analysis,electron microscopy and Western blotting analysis.After 10 generations of recombinant virus,the fluorescence produced no change.Electron microscopy showed that the shape and structure of two recombinant virus consistent with the parental virus with no change.Western blotting analysis showed that,two recombinant virus expressed VP60 protein,sizes were about 60 ku,can react with RHDV hyperimmune serum.The recombinant protein had good reactogenicity.It suggested that two SWPV recombinants have the potential as RHDV vaccines for the use in rabbits.