ABSTRACT
Objective To detect the immune effect of FbaAmAb2 against the surface protein of group A Straptococcus (GAS),and explore the pathogenesis and therapy of GAS infections.Methods By subclonal and bacterial ELISA,the positive hybridoma cells were screened that can produce better titers of FbaAmAb2 against GAS-surface FbaA protein,and were injected into the peritoneal cavities of BALB/c mice to produce ascites.The collected ascites were performed to dilute,as follows,original ascite,1:2,1:4,1:8,and 1:16 to test tube agglutination.Based on the results,we selected appropriate dilution to passively immunize mice,and then challenged the mice with GAS,evaluating FbaAmAb2 neutralizing ability with GAS in mice by the survival rate of the immunized mice.Whether FbaAmAb2 could inhibit the binding of factor H to GAS was confirmed by the invasive inhibition assay.Results The IgG titer of bacteria solution ELISA is 1:160 and the titer of tube agglutination is 1∶8.The protect rates of FbaAmAb2 on preventing mice with GAS infections are as follows:66.67％ in original ascite and 1:2 diluted groups,and 50％ in 1:4 diluted group.Mice in each experimental group were evoked significantly protective immune responses compared with the PBS control by SPSS analysis.FbaAmAb2 can competitively inhibit factor H binding to the surface proteins FbaA of GAS,which decreased the entry of GAS into the cytoplasm of human epithelial cells through the binding of factor H.Conclusion FbaAmAb2 is promising to be used in emergent prevention or the clinical therapy for GAS infection and it is promising starting points for pharmacologic targeting and further development of new therapeutic agents for GAS.
ABSTRACT
Objective:To identify monoclonal antibody McAb2 recognizing epitope of Fba of GAS.Methods:The overlapped peptides were synthesized and their abilities to bind McAb2 were detected by dot-ELISA.The predominance amino acids specific for McAb2 were screened using phage 7 peptide library.Results:The result by dot-ELISA analysis demonstrated that the synthetized peptide,amino-acid residues 100-112~(th),could bind McAb2 with high affinity.The predominance amino acids specific for McAb2 were ITPDL,which was located in 100-110~(th)aa of Fba by panning with phage 7 peptide library.Conclusion:The domain and the predominance amino acids of Fba recognized by McAb2 is determined.The results would contribute to the research of the role of Fba on the pathogenic mechanism of GAS,the identification of function of McAb2,and the development of epitope-peptide vaccine.
ABSTRACT
Objective:To construct eukaryotic expression plasmid ecording a novel surface protein Fba of GAS, and to explore its impact on host immune responses.Methods:Fba gene was amplified by PCR using strain SSI-9 (GAS M1 serotype isolates) as the template, then cloned into pcDNA3.1 for constructing eukaryotic expression plasmid pcDNA3.1/fba and sequenced. Female CD1 mice were randomly individed into 6 groups, and immunized respectively with Fba protein, M protein, pcDNA3.1/fba + Fba protein, pcDNA3.1/fba, pcDNA3.1 and PBS as control. Blood was obtained from the mice and specific antibody of IgG was detected by ELISA. Spleen cells were assessed with lymphocyte proliferation assays.CD4+T cell and CD8+T cell were detected by flow cytometry (FCM). Assay results were analyzed with SPSS10.0.Results:The IgG against Fba protein kept hightest levels in group immunized with Fba protein. The levels of lymphocyte proliferation, CD4+, CD8+T cell were significantly high in the group pcDNA3.1/fba. Conclusion:(1) Just as M protein, the antibody to Fba protein could be efficiently induced by immunization with Fba protein, which showed that Fba protein was hopefully to be a candidate protein for vaccine against GAS.(2) Eukaryotic expression plasmid pcDNA3.1/fba was successfully constructed and this recombinant plasmid could efficiently induce antibody and CD4+ T cell for againsting GAS.