Expression And Immunogenicity Of Staphylococcus Aureus GapC Protein

Staphylococcus aureus (S. aureus) is an important pathogen that causes a varitety of diseases among human and animals. Due to the lack of common protective antigens among the S. aureus isolates, existing vaccines available remain ineffective in clinical trials. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein can be used as antigens in vaccines to protect against parasitic and microbial infections. Thus, the object of this study is to use conserved S. aureus surface antigen GapC, which with the GAPDH activity, as the basis for an immunogen, and to evaluate its immunogenicity and potential to protect against S. aureus infection.Firstly, gapC gene of S. aureus strain BMSA/855/23-1 was amplified by PCR, and was introduced into pMD18-T vector subsequently. The recombinant clone, pMD-T/gapC, was sequenced. The nucleotide sequence of gapC was compared and analyzed with the one published previously in GenBank, and then, pQE30/gapC recombinant plasmid was constructed by inserting gapC gene into pQE-30 vector. The recombinant plasmid was transformed into E. coli strain M15 (pREP4), and was induced with IPTG to express recombinant GapC fusion proein. The expression of target protein was detected by SDS-PAGE. The GAPDH activity assay and Western blot assay were performed to the purified recombinant protein thereafter. To characterize the antigenicity of the GapC protein, 25 healthy rabbits were randomly devided into 5 groups, immunized with the recombinant protein, killed S. aureus vaccinum and placebo (PBS) respectively. Enzyme-Linked Immunosorbnent Assay (ELISA) was used to measure the levels of IgG antibody titres and the concentration of IFN-γand IL-4 cytokines in rabbit serums. Finally, the immunized rabbits were challenged with the S. aureus strain WOOD46, clinical symptoms, body temperatures and pathological changes were observed to characterize the morbility of the challenged rabbits, which was then comfirmed by the isolation and identification of the S. aureus from livers, spleens and kidneys organs. The immunoprotection of the recombinant GapC proein was evaluated accordingly.The results of the study showed that 1008bp lengthed gapC gene of S. aureus strain BMSA/855/23-1 was successfully amplified and cloned. Sequencing results showed that the gapC gene of the isolated strain shares 99.3% homology in nucleotide sequence, and 99.4% in amino acid sequence with that of the S. aureus strain BM10 in GenBank. SDS-PAGE result showed that the recombinant GapC was expressed successfully in E. coli M15(pREP4)strain, the high GAPDH activities and antigenicity were comfirmed by GAPDH activity assay and Western Blot. The IgG antibody titres against GapC and whole cell in rabbit serums reached its peak at day 28 after boost immunization (1:64000). The concentration of IL-4 and IFN-γin GapC groups rabbit serums increased significantly (p<0.05) at day 14 after boost immunization, while the concentration of those in whole cell group did not increase (p>0.05) compared with the contol. Only 1 rabbit in 5 of the protein immuned group showed clinical symptoms and higher body temperatures after challenged with 5×107CFU S. aureus.From the results above, it can be concluded the expressed recombinant GapC protein had high GAPDH activity and immunogenicity;The GapC protein can also protect against S. aureus challenge to some extent. These results suggest that S. aureus GapC protein could be an attractive target for further genetic engineering vaccine.