The Research And Identification To The Epitopes Of The Monoclonal Antibody To Fba Of GAS

Objective: Group A streptococcus (GAS) is a common bacterial pathogen of human which can cause serious invasive disease and post-infection autoimmunity disease. Penicilin has been the choice against streptococcus, however, because of the increase of the drug-resistence bacteria stains and the immune escape, some infections caused by GAS were not able to be cured yet, and people would be infected by the pathogen repeatly. It is still an effective method to induce higher titer of antisera which can provide effective protection agaist GAS infections. While the effective vaccine to GAS is not produced until now, for it contains epitopes known to cross-react with tissues in several animals, including humans. Besides, it possesses multiplicity Serotypes [1].A novel protein, Fba, an Fn-binding protein expressed on the surface of GAS, was reported by Terao in 2001[2]. It exists in many serotypes and shows a high homology among different serotypes. Since Fba belongs to Fn(Fibronectin,Fn) binding protein, Fba, via the binding of Fn, also mediates the internalization of GAS to Hep-2 cells. It reveals that Fba potentially promote the entry of GAS into human epithelial cells to shield the bacterium from antibiotics and the immune system[3]. Furthermore, Fba can bind complement regulator protein FH and FHL-1, which can promote the escape of the complement attack and contribute the anti-phagocytosis[4,5]. Our former researches suggest that Fba shows strong immunogenicity and can induce protective immunization against GAS[6,7]. Thus, Fba protein is considerd to be a candidate vaccine of GAS.The study on Fba in our laboratory has been going on till now, especial for the role in the escape of immunological attack. Two cell lines secreting antibodies against Fba were obtained, and the secreted monoclonal antibodies (McAb) were designated as McAb1, McAb2 respectively. McAb2 was found to be able to bind wide-type GAS with affinity, in addition, McAb2 was found to be able to just bind truncated Fba68-161by ELISA and Western blot. The corresponding epitope of McAb2 was supposed to contain at least the 104~108thaa of Fba according to experiment results. Based on the specific binding domain of McAb2, bioinformatics was used to predict the corresponding epitope of McAb2, subsequently the fusion proteins which contained or deleted the predicted epitope were expressed respectively by genetic method. Meanwhile we utilized the phage peptide library to filter the biding situs of McAb2. Whether the biding situs coincidence with the binding situs of FH to Fba was detected by ELISA and plasma adsorption experiments. The results are able to facilitate 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.Methods:1 According the binding domain of McAb2 to Fba, The corresponding epitope was predicted through bioinformatics, and three overlapped epitopes, which contained all or part of the presumed epitope, were consided. The genes were amplified by PCR,and then expressed in E.coli.,subsequently, the ability of fusion proteins binding to McAb2 was verified by Western blot.2 The fba gene, which was deleted the corresponding gene of the presumed epitope, was amplified by PCR and expressed in E.coli. Whether McAb2 was specific for the mutant Fba protein was detected by Western blot and ELISA.3 The overlapped peptides were synthetized and their abilities to bind McAb2 were detected by dot-ELISA.4 The predominance amino acids specific for McAb2 were filtered using phage 7 peptide library.5 Whether McAb2 could compete with FH to bind Fba was detected by competive ELISA.6 Whether McAb2 could prevent FH of human blood plasma to bind Fba was detected by plasma adsorption experiments.Results:1 Three prokaryotic expression plasmids of the predicted gene fragments, pGEX-4T-2/fba84-101, pGEX-4T-2/ fba95-114,pGEX-4T-2/fba101-118,were constructed and expressed successfully. Western blot analysis showed that Fba95-114 and Fba 101-118 can significantly bind McAb2.2 The prokaryotic expression plasmid of the gene deleted the corresponding epitope, pGEX-4T-2/fba68-161â–³96-118, was constructed and induced to express successfully. Western blotting analysis showed that McAb2 can not bind Fba 68-161â–³96-118 , which was coincident with Indirect ELISA.3 The synthetical-overlapped peptides were the amino- acid residues 100~112th, 104~116th and 108~120th of Fba. The result by dot-ELISA analysis demonstrated that the synthetized peptide, amino-acid residues 100~112th, can bind McAb2 with high affinity.4 The predominance amino acids specific for McAb2 were ITPDL, which was located in 100~110thaa of Fba by filtering in phage 7 peptide library.5 McAb2 can partly block the binding of FH with wild-type GAS by ELISA analysis.6 Plasma adsorption experiments demonstrated that wild-type GAS can bind FH in human plasma, however, McAb2 can inhibit FH to bind Fba.Conclusions:1 Peptide specific for McAb2 and protein deleted the predicted epitope were expressed successfully. The domain where the epitope of McAb2 located was determined primaryly. 2 The predominance amino acids of McAb2 were measured by phage 7 peptide library.3 McAb2 can prevent or at least partly prevent FH to bind Fba, which facilitated to identify the function of McAb2 and to research the pathogenic mechanism of GAS.