Expression And Immunogenicity Of Staphylococcus Aureus IsdB Protein

Staphylococcus aureus (S. aureus) is one of important pathogen that causes a variety of diseases among human and animals. S. aureus can utilize the host hemoproteins as iron sources for bacterial growth. IsdB is a hemoglobin receptor required for iron utilization and is highly conserved in S. aureus. Research shows the protein is highly immunogenic in mice and rhesus macaques and produces antibody levels is significantly higher than other surface proteins. Therefore, studying immunogenicity of the IsdB protein, using the mechanism of IsdB-mediated scavenging of iron from hemoglobin for developing human and animal vaccines against S. aureus will have important value in practice and theoretical significance.Firstly, isdB genes of 50 strains of S. aureus for bovine mastitis were amplified by PCR to determine the conservatism of IsdB in S. aureus for bovine mastitis in China. Then, isdB gene of S. aureus strain BMSA/855/23-1 was ligated into pMD18-T vector. The recombinant clone, pMD18-T-isdB, was sequenced. The nucleotide sequence of isdB was compared and analyzed with the one published previously in GenBank. Further the isdB fragment was ligated into pQE-30 expression vector. The positive recombinants was named as pQE-30-isdB.Then, it was transformed into E.coli XL1-Blue and was induced with IPTG to express recombinant IsdB fusion protein. The expression of target protein was detected by SDS-PAGE.Immunize mice with the purified recombinant protein and killed S. aureus vaccinum. Western blot assay were performed to characterize the antigenicity of the IsdB protein. Establish the ELISA method to detect the IgG antibody titres against IsdB and whole cell in mouse serums. 13 strains of S. aureus from mastitis were detected with IsdB immune serum in microagglutination test. MTT and ELISPOT detected T lymphocyte transformation effect and spleen lymphocytes cytokines IFN-γlevels of protein group mice at the tenth day after strengthen immunization. At the fourteenth day after booster immunization, the immunized mice with IsdB were challenged with different S. aureus strains and the immunized mice with killed S. aureus vaccinum were challenged with the S. aureus strain BMSA/855/23-1. The immunoprotection of the recombinant IsdB protein was evaluated accordingly.The results of the study showed that isdB gene of all 50 strains was amplified, proved that isdB gene was highly conservative in S.aureus for bovine mastitis in China. Sequencing results showed that the isdB gene of the isolated strain shares 98.8% homology in nucleotide and amino acids sequence with that of the S. aureus strain MW2 in GenBank. SDS-PAGE result showed that the recombinant IsdB was expressed successfully in E. coli XL1-Blue. Western blot assay indicated that the protein had high antigenicity. The ELISA results showed that the IgG antibody titres against IsdB and whole cell in mouse serums reached its peak at the third week after boost immunization (1:64 000). The antiserums showed a specific immunological response to standard antigen of 12 strains of S. aureus in microagglutination test, proved that IsdB antiserum has the antigen-antibody reaction capacity with different isolates. The T-lymphocyte transformation test with MTT assay results compared with the control group the difference was significant (p<0.05) and ELISPOT results compared with the control group was significantly increased (p<0.05), shows that IsdB can stimulate mice to produce better cell-mediated immune response. The protection of IsdB for S.aureus BMSA/855/23-1 strain rate of 70%, and SH-12 protection rate of 80%, and SH-16 protection rate of 50%, the whole cell group BMSA/855/23-1 protection rate of 80%.From the results above, it can be concluded isdB gene was highly conservative in for cow mastitis in China. The expressed recombinant IsdB protein had high immunogenicity and immunoprotection efficiency. These results suggest that S. aureus IsdB protein could be applied into prevention for S. aureus infections.