Identification And Characterization Immunogenic Proteins Of Nocardia Farcinica IFM 10152

Nocardia species produce cutaneous,pulmonary and central nervous human and animal diseases.Nocardia farcinica is one of the most commonly isolated species.To uncover virulence factors,early diagnostic reagents and effective vaccines,cellular proteins and secreted proteins of N.farcinica IFM 10152 were analyzed using an immunoproteomics method.In this study,the cellular proteins and secreted proteins of N.farcinica IFM 10152 were extracted to perform two-dimensional polyacrylamide electrophoresis.The gels were used for stained or reacted with immune rabbit sera to screen immunogenic proteins by Western blot.These antigenic proteins were removed from the gel and digested with trypsin.Tryptic peptides subjected to matrix-assisted laser desorption ionization mass spectrometry（MALDI-TOF-MS）to identifiy proteins.A total of 13 proteins were identified among 19 proteins spots by MALDI-TOF-MS.Among these,5 proteins（NFA₄5140,NFA₅55680,NFA₄8660,NFA 49580 and NFA₁5900）were expressed in E.coli and antigenicity was analyzed with Western blot.To explore the immunological characteristics of these 5 proteins,mice were immunized with recombinant protein,respectively.The levels of IgG,IgG1 and IgG2a to NFA₄5140,NFA₅55680,NFA₄8660,NFA₄9580 and NFA₁5900 were measured by indirected enzyme-linked immunosorbent assays（ELISAs）.The results showed that all five proteins elicited a potent humoral immune response with high concentration of specific antibodies,particularly IgG1 which has been proved to be involved in T-helper cell type Ⅱ（Th2）-immune responses.The cytokines（IFN-γ,IL-2 and IL-4）in the culture supernatants produced by splenocyte isolating from the immunized mice and stimulated with recombinant protein were detected.An increase was observed in the levels of IFN-y,IL-2 and IL-4.The results suggested these five proteins elicted both cellular and humoral immune responses in an infected host.To understand the role of these five proteins,the epitope,amino acid homology analysis,function and virulence were predicted by bioinformatics approach.The results showed NFA₄8660、NFA₄9580 and NFA₁5900 were conserved among pathogenic Nocardia but none were for M,tuberculosis,suggesting that they may be the clinical diagnosis markers.NFA₅5680、NFA 49580 and NFA₁5900 were identified as "virulent proteins" and "protective antigens" which may cause bacterial virulence and protective immunity.In summary,a total of 13 novel immunogenic proteins of N.farcinica were indentified and we have evaluated immunological value of 5 proteins among these.They may contribute to lay a molecular foundation for clarifying pathogenic mechanism and provide new candidate molecules for development of diagnosis marker and new vaccine for N.farcinica.