Immunoprotection Of Staphylococcus Aureus IsdA Protein

Staphylococcus aureus (S. aureus) is an important human and animal pathogen. S. aureus is found on the skin and mucosal surfaces of humans and cause a variety of important diseases of animal. As cross-protective immunity of different S. aureus strains is limited, the present study of S. aureus vaccine in clinical are not ideal. S. aureus iron-regulated surface determinant (Isd) is essential for the survival of bacteria, and IsdA play a key role in this system. IsdA, the surface protein of S. aureus, was prokaryotic expressed and prepared immunogen, to explore the protective effects of immunization of IsdA.Firstly, isdA gene of S. aureus strain Newman was amplified by PCR, and was ligated into pMD-18T vector and sequenced, sequencing results compared with the one published previously in GenBank. Construction of the pET32a-isdA prokaryotic expression vector, it was expressed in Rosetta (DE3) after induced with IPTG. The recombinant IsdA fusion protein was detected by SDS-PAGE. Western blot assay were performed to characterize the immunogenicity of the recombinant IsdA protein. At the same time, the recombinant IsdB protein was expressed and purified, to compare the protective effects of immunization of IsdA protein. Then, 120 healthy mice were divided into 12 groups for the IsdA protein group, IsdB protein group, IsdA + IsdB protein group, non-immunized control group. Each group protein was emulsified with Freund's complete adjuvant and Freund's incomplete adjuvant for preparation of immunogen. Other twelve healthy mice were divided into 4 groups, immunized by the same method to measure the antibody titer. Every group for getting serum was taken serum every week and IgG antibody levels in serum were detected by ELISA. IFN-γ, IL-4 cytokines levels in serum that collected in the first week after the frist immunization were detected by ELISA. The second immunization begins the third week after the first immunization, and challenged by S. aureus strains Wood46, Newman, HLJ855-23-1 in the second week after the second immunization, record the death for one week.Immunity test results show that the expressed recombinant protein IsdA + IsdB combined immunization have a preferable immunoprotection. By the detection of cell immunity with cytokines IFN-γand IL-4, and the IgG antibody levels, the test results show that the IsdA + IsdB combined immunization can effectively stimulate cellullar immunologic response and humoral immunoresponse in mice, and the response level is significantly higher than the control group and the protein immunization alone.