The Preparation Of Monoclonal Antibody To Fba Of GAS And The Primary Identification Of Its Epitope Of The Obtained Antibody

Objective: Group A streptococcus (GAS) is a common bacterial pathogen of human which can cause the throat, skin, and soft tissue infection. It is also a cause of children toxic diseases such as scarlet fever, toxic shock symptom. Meanwhile the harms induced by GAS exist in the post-infection autoimmunity disease, for example rheumatic fever, rheumatic heart disease, glomerulonephritis, psoriasis, immunity cerebrosis etc. However, Some Group A streptococcus infection still can't be cured and people can be infected repeatlly, because of the occurrence of the drug-resistence bacteria stains and the immune escape.Vaccine is still the effective method to prevent and control the infection of the streptococcal infection. In spite that the streptococcal vaccine research can date from forty years ago, perfect vaccine is not produced yet for many reasons. So far people pay more attention on M protein as a vaccine candidat. However, because of the variety of the serotype and cross-reaction of M protein with many human organisms, for example, heart, kidney, articular cartilage, collagen tissue, cerebra basal ganglia, etc. The research for non-M protein vaccine never stoped, the different study refer to the F1 protein, Sse protein, C5peptase, ectotoxin and so on. Nevertheless all the immune effection can't surpass the M protein. Therefore combining the M protein and non-M protein is becoming a tendency.Fba protein (Fibronectin-binding protein a) is a new streptococcus surface protein found by Terao in 2001. It shows a high similarity (69%) to the Fn-binding protein of S. aureus FnBPA. The gene is designated as Spy2009. The fba gene was found to consist of 1068 nucleotides and encodes a protein with 355 amino acids and calculated molecular mass of 37.8KDa. The whole protein contains the putative signal-peptide region,α-helical coiled coil regionm, proline-rich repeats region, C-terminal cell wall anchor region. It exists in many serotypes such as M1, 2, 4, 9, 13, 22, 28, 44, 49, 60, 67, 75, 77, 79, 80, 82, 87 and 89 serotypes. Among different serotype, Fba protein shows a high homology. The results of researche demonstrate GAS expressing Fba stain (Fba+ GAS) can invade the human epithelial cell more easily than the GAS stain not expressing Fba. It can bind complement regulator protein FH, FHL-1, which can promote the escape of the complement attack and contribute the anti-phagocytosis. Our former researches reveal that Fba can induce same protective immunization as M protein to prevent the streptococcal infection.The research aim to produce the monoclonal antibodies of Fba, express the truncated protein and analyze the binding domain of the monoclonal antibodies produced. The result can facilitate the research of the function of Fba and the production of epitope-peptide vaccine.Methods:1 BALB/c mice were immunized with GST-Fba recombination protein accompanied with adjuvant. The titer of antiserum was tested by indirect ELISA using GST-Fba recombination protein as the coated antigen. The spleen cells of the immunized mice were hybridized with the myeloma cell SP2/0, and the hybridoma cells were selected by HAT culture medium. The positive hybridoma cells secreting the antibody against Fba were primary detected by the indirect ELISA, then subcloned via limiting dilution. Positive hybridoma cells were injected into the abdominal cavity of BALB/c mice to produce antibodies.2 Analyse the characters of monoclonal antibody. The titers of monoclonal antibody ascites and supernament were measured by indirect ELISA using GST-Fba as coated antigen. The specificity of monoclonal antibody was analyzed by Western blotting. In order to identify whether the linear antigenic epitopes that monoclonal antibodies could bind to were natural epitope located on the surface of the GAS, whole GAS was cultured and coated plates to test the ability of binding McAb.3 Truncated Fba proteins which were divided into four overlaps based on the structural domains were designed. The amino acid residues in 4 overlap peptides are 37~110aa, 68~161aa,104~277aa,160~324aa respectively. They were designated as FbaA1,FbaA2,FbaA3,FbaA4 accordingly.4 The fba gene was amplified by PCR, and cloned into prokaryotic expression plamid pGEX-2T. Sequencing showed the plamid corrected. The plasmids were transferred into E.coli BL21 and induced with IPTG to express truncated Fba proteins. The products were analyzed by SDS-PAGE electrophoresis and confirmed by Western blotting. The truncated proteins were purified by affinity chromatography column.5 The domains of truncated Fba protein that monoclonal antibodies could bind were identified by Western blotting and ELISA.6 The epitopes of Fba protein were predicted with sofeware online.Results:1 Two cell lines secreting antibodies against Fba were obtained through three times subcloning successfully. The monoclonal antibodies obtained were designated as McAb1, McAb2 respectively.The fusion rate was 37.1%.The positive colon rate is 2% by GST and GST-Fba proteins selection.2 The titer of the specific antibody in supernatant and ascites were tested by ELISA. The highest titer was 1:6.4×102 and 1:105 respectively. The results of Western blotting demonstrated that McAb could specifically bind with GST-Fba, but not with GST. ELISA using whole bacteria as coated antigens revealed that McAb1 could bind both Fba+ GAS and Fba- GAS stains, demonstrating that the epitope binding McAb1 may not exist on the bacterial surface or is not a natural epitope, or possible that the McAb1 have cross-reaction with other surface proteins of GAS. McAb2 could bind Fba+ bacteria only, so McAb2 may be one special antibody of Fba, the corresponding epitope may just be a natural linear epitope of Fba locating on the surface of GAS.3 Productions of PCR were matched with length designed, which were 242bp, 302bp, 540bp, 512bp respectively.4 Four truncated GST-Fba proteins were expressed under induced by IPTG with SDS-PAGE analysis. Western blotting analysis indicated that the recombinant proteins could be recognized by GST McAb.5 McAb1 could bind truncated proteins FbaA3 and FbaA4, while McAb2 could only bind FbaA2 by Western blotting analysis. The same results were showed by ELISA as Western blot. The epitope corresponding McAb2 were supposed to span the 104-110aa according experimental evidences.6 The experimental results were identical with prediction of the B-cell epitope online.Conclusions:1 Two cell lines secreting monoclonal antibodies against Fba were obtained. The biological characteries were identified. This provides a convenient tool for further reseach on the function of Fba protein. 2 Four truncated Fba proteins were expressed successfully and domains of Fba corresponding the monoclonal antibodies were identified primally. The results could contribute to the future research on the function of Fba, and epitope-peptide vaccine.