Construction and identification of recombinant swinepox virus expressing p72 protein of African swine fever virus
[Objective]The objective of this study was to construct a recombinant swinepox virus that can efficiently express the p72 protein of African swine fever virus,thus exploring the feasibility of swinepox virus as a new live vaccine vector for African swine fever.[Method]In this study,the ASFV B646L gene was synthesized and cloned into the universal vector pUSG11/P28 to generate the recombinant plasmid pUSG11/P28-B646L.The plasmid pUSG11/P28-B646L was transfected into PK15 cells pre-infected with swinepox virus by lipofectamine to generate recombinant virus rSPV-B646L.The recombinant virus was screened and purified after the lesion appeared.The recombinant virus was identified by PCR and gene sequencing,the expression of p72 protein was detected by Western blotting and indirect immunofluorescence assay,and the proliferation characteristics and genetic stability of the recombinant virus were determined.[Result]The results showed that swinepox virus recombined successfully with the plasmid pUSG11/P28-B646L in PK15 cells,and the purified recombinant virus contained B646L gene which had a correct gene sequence.The results of Western blotting and indirect immunofluorescence assay showed that recombinant virus rSPV-B646L could express p72 protein in PK15 cells,which showed correct size and had a specific reaction with p72 monoclonal antibody.The one-step growth curve showed that there was no significant difference in the proliferative characteristics between the recombinant virus and the parental virus.After the recombinant virus was passed on PK15 cells continuously for 15 generations,there was no mutation or deletion of the inserted foreign gene.[Conclusion]In this study,we constructed successfully a recombinant virus rSPV-B646L by using swinepox virus as a carrier.The recombinant virus had good genetic stability in PK15 cells.The insertion of B646L gene did not affect the proliferation ability of the recombinant virus in PK15 cells,and the expressed p72 protein had reactogenicity.The results of this study laid a foundation for the development of recombinant SPV vector vaccine for ASF.
African swine fever virusrecombinant swinepox viruslive vaccine vectorp72 protein
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