Construction of a recombinant Bacillus subtilis strain expressing SpaA and CbpB of Erysipelothrix rhusiopathiae and evaluation of the strain immunogenicity in a mouse model
To construct a recombinant Bacillus subtilis strain expressing SpaA and CbpB of Erysipelothrix rhusiopathiae for oral administration,we constructed the recombinant plasmid pDG1730-CBJA by fusion PCR and seamless cloning.The plasmid was introduced into B.subtilis KC strain by natural transformation,and the recombinant strain KC-spaA-cbpB was screened out on the plate containing spectinomycin(sper)and confirmed by PCR and starch degradation test.The SpaA and CbpB expressed by KC-spaA-cbpB were detected by Western blotting and indirect immunofluorescence assay,and the genetic stability of the recombinant strain in mice was determined.The plasmid pMAD-∆sper with knockout of sper was constructed and transformed into KC-spaA-cbpB.The sper-deleted mutant strain KC-spaA-cbpB::∆sper was screened and identified,and its immunogenicity in a mouse model was evaluated by oral immunization.The results showed that the recombinant strain KC-spaA-cbpB was stable in mice,expressing SpaA on the cell surface and CbpB on the spore surface.KC-spaA-cbpB::∆sper expressed SpaA and CbpB.The mice vaccinated with the spores of KC-spaA-cbpB::∆sper had higher levels of SpaA and CbpB-specific IgG in the serum that those vaccinated with the wild-type spores 42 days after vaccination by gavage(P<0.01).The protective rate of mice immunized with the recombinant spores was 67.5%.The results indicated that a recombinant B.subtilis strain expressing SpaA and CbpB of E.rhusiopathiae was successfully constructed,and the recombinant strain laid a foundation for the development of oral live vector vaccines for swine erysipelas.
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维普
