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 (spe^r) 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-∆spe^r with knockout of spe^r was constructed and transformed into KC-spaA-cbpB. The spe^r-deleted mutant strain KC-spaA-cbpB: : ∆spe^r 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: : ∆spe^r expressed SpaA and CbpB. The mice vaccinated with the spores of KC-spaA-cbpB: : ∆spe^r 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.
Animals ; Bacillus subtilis/immunology* ; Mice ; Erysipelothrix/immunology* ; Bacterial Proteins/immunology* ; Bacterial Vaccines/genetics* ; Erysipelothrix Infections/prevention & control* ; Immunization ; Mice, Inbred BALB C ; Plasmids/genetics* ; Immunogenicity, Vaccine ; Administration, Oral ; Antigens, Bacterial

Spore surface display is one of attractive microorganism surface display systems. With the advantage of resistance attribute and specific assembly pattern, the technology of spore surface display now is attracting more and more attention. According to the current reports and main achievements of spore surface display, the structure and assembly of spores, the principle for construction and some existing spore surface display systems were elaborated in this paper. Now with the unique property of spores, the technique is not only widely used in production of vaccines but also has great applied potential in the field of biocatalysis and cell-factory.
Bacillus subtilis ; genetics ; metabolism ; Biocatalysis ; Biotechnology ; methods ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; methods ; Recombinant Proteins ; genetics ; metabolism ; Spores, Bacterial ; genetics ; metabolism ; Tetanus Toxoid ; genetics ; immunology

We amplified dhbC gene from the siderophore producing bacterium CAS15 by PCR. After ligated the PCR product to pMD18-T vector and then sequenced, we obtained a 1197 bp fragment. The blast result showed that the nucleotide acids of dhbC gene (Accession No. FJ194456) of CAS15 shared 99.7% identity with that of dhbC gene of Bacillus subtilis (GenBank Accession No. Z99120), and was predicted to encode a 43.8 kD polypeptide with 398 amino acid residues. We cloned the dhbC gene into expression vector pET-30a(+) and then transformed into Escherichia coli BL21(DE3) via calcium chloride transformation method, and obtained the recombinant E. coli BL21(DE3)/pET-30a-dhbC. Induced by 1 mmol/L IPTG the fusion protein 6His-DhbC, a 48.8 kD polypeptide was successfully expressed mainly in soluble form in E. coli BL21(DE3), and the amount reached highest at 30 degrees C for 4 h. According to the N-terminal fusion 6 His-tag, we purified the recombinant polypeptide by Ni2+ metal affinity chromatography and finally identified it by Western blotting. The result indicated that the recombinant DhbC had the antigenicity to rabbit anti-his-tag polyclonal antibody, which provides the basis for the study of practical utilization in production and the biocontrol mechanism of B. subtilis. Finally, we deleted dhbC gene by gene knockout and then retransformed it into the dhbC gene-delected mutant, which confirmed that dhbC gene play an important role in siderophore biosynthesis.
Bacillus subtilis ; enzymology ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Hydrolases ; genetics ; metabolism ; Hydroxybenzoates ; metabolism ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Siderophores ; metabolism