Immunogenicity Analysis Of Surface Protein Fba From Streptococcus Pyogenes

Objective: Streptococcus pyogenes (GAS) is a widespread human pathogen causing infections ranging from superficial infections of the throat or skin to highly invasive and potentially lethal infections. These diseases are initiated by the adhesion of GAS to epithelial cells in the respiratory tract or skin. Interactions between GAS surface molecules and epithelial cell receptors are mediated by fibronectin in serum, which can promote the entry of streptococci into the cytoplasm of human epithelial cells, so GAS can evade ingestion or killing by host phagocytes[1].Fba is a recently identified GAS cell surface protein capable of binding FHL-1, FH and fibronectin, and present in at least 18 GAS serotypes. It was demonstrated that Fba contributes to GAS resistance to phagocytosis and, via binding to FHL-1, can promote the entry of streptococci into the cytoplasm of human epithelial cells[2, 3]. In the study, we had cloned Fba gene into plamid for constructing the prokaryotic expression plamid and the eukaryotic expression plamid. And the combinant proteins were expressed. The expression of protein fba was confirmed by Western blot analysis and ELISA .To study the immunogenicity, explore the immunity to infection of GAS and to provide the foundation for the research on diagnostic reagent and vaccine, mice were injected with pcDNA3.1/fba and Fba protein ,after last boost detected its'cellular and humoral immune responses.Methods: 1 Fba gene was amplified by PCR and cloned into pMD18-T vector for sequencing. The gene was digested with BamHI, EcoRI and BamHI, XhoI, and then cloned into prokaryotic expression plamid pGEX4T-2 and eukaryotic expression plasmid pcDNA3.1.2 The protein Fba induced by IPTG was high-expressed in E.coli as inclusion body, and the protein Fba was purified dire- ctly from SDS polyacrylamide gel by electrodialysis. The cha- racter of the recombinant protein was analysed by ELISA and Westernblot.3 Purified Fba protein as antigen was coated on the microt- iter plates to detect the sera from mouse infected by GAS and the sera from patients of GAS infection. Purified M protein of GAS as positive control was did same as positive control.4 Female BALB/c mice were randomly individed into 6 groups, and immunized respectively with Fba protein, M protein, Fba pcDNA3.1/fba + protein, pcDNA3.1/fba, pcDNA3.1 and PBS as control. Blood was obtained from mice tail and the sera IgG was detected by ELISA. Spleen cells were obtained, and CD4+T cell and CD8+T cell were detected by flow cytometry (FCM). Mice immunized with Fba protein and plasmid were challenged with GAS to evaluate its protection. Results: 1 Fba gene was amplified by PCR, after sequ- encing cloned into pcDNA3.1, pGEX4T-2. Prokaryotic expr- ession plamid pGEX4T-2/fba, and eukaryotic expression plasmid pcDNA3.1/fba were successfully constructed.2 The protein Fba was high-expressed in E.coli as inclusion body. The experiment results of ELISA and Westernblot showed the recombinant protein had a good affinity with anti-Fba sera of rabbit.3 The purified recombinant Fba protein was able to react with anti-GAS sera of mouse, and sera of patients by ELISA. The results were consistent, although the OD405 value tested with recombinant Fba protein as coated antigen was less than that with recombinant M protein.4 The IgG to Fba protein kept hightest levels in the Fba protein immunized group, pcDNA3.1/fba+Fba protein, pcDNA3.1/fba immunized group followed it. The levels of lymphocyte proliferation and CD4+, CD8+T cell were higher in the group pcDNA3.1/fba than that in the others.5 Challenged with GAS, mice immunized with PBS were all dead in 4 days, and a protection rate of 60% was observed for mice immunized with Fba protein, pcDNA3.1/fba+Fba protein or M protein in 14 days, the survival rate for mice immunized with pcDNA3.1/fba was 40%.Conclusion: 1 Eukaryotic expression plamid pcDNA3.1/ fba and prokaryotic expression plamid pGEX4T-2/fba were successfully constructed. 2 Fba protein had been high-expressed in E.coli as recombinant protein. GAS infection was able to induce anti-Fba protein and anti-M protein sera, which revealed that the Fba protein as same as M protein have a good immunogenicity and antigen specificity. As we known, M protein binding its antibody were the major cause of the hypersensitivity after human infected GAS, addition to the high diversity of the its serotype, M protein as the candidate of GAS vaccine was restricted. So Fba protein can be taken into account as the candidate of GAS vaccine, also as diagnostic antigen.3 Humoral immune responses to GAS could efficiently induced by Fba protein. Fba eukaryotic expression vector efficiently induced humoral responses and Th responses against GAS, and mice immunized with Fba protein or plasmid gained ability against GAS infection, which showed that Fba protein was hopefully to be a candidate for vaccine against GAS.