A族链球菌表面蛋白Fba单克隆抗体的制备及其对应表位的初步鉴定

Preparation of monoclonal antibodies against Fba protein of Group A streptoccus and primary identification of the corresponding epitopes

目的:制备抗A组溶血性链球菌(GAS)表面蛋白Fba(fibronectin-binding proteins a)的单克隆抗体(mAb),并对单抗的生物学功能及其相应表位进行了初步鉴定。方法:以重组Fba蛋白为免疫原免疫BALB/c小鼠,运用杂交瘤技术及间接ELISA筛选出针对Fba的杂交瘤细胞株,以Western blot鉴定mAb的特异性,并将获得的mAb与Fba+GAS和Fba-GAS分别行全菌ELISA、与Fba的分段表达蛋白行Western blot,初步鉴定mAb针对的表位所在区域。结果:获得了稳定分泌抗Fba-mAb的杂交瘤细胞株,其中一株mAb能特异结合天然Fba+链球菌,且该单抗针对的表位包含了Fba蛋白的第104～108位氨基酸,该位点也是链球菌Fba蛋白结合补体调节蛋白FH的位点。结论:成功地制备了抗GAS表面Fba蛋白的mAb,其中一株mAb对应的表位为位于菌体表面的天然表位,并检测出了该mAb对应的表位所在区段,这为深入研究GAS的致病机制提供了有力工具。

Objective： To prepare monoclonal antibodies against surface-associated fibronectin-binding proteins （Fba） of Group A streptococcus （GAS） and perform immunological identification of the monoclonal antibodies. Methods： BALB/c mice were immunized with recombinant Fba proteins to prepare immunized spleen cells, and the hybridomas were obtained with hybridoma technique. Specificity of the monoclonal antibodies was analyzed by western blot, and the binding domains of trtmcated Fba protein were identified with acquired mAb by western blot and ELISA. In order to determine whether the corresponding epitopes of the monoclonal antibodies were natural epitopes located on the surface of GAS,Fba^＋ GAS or Fba^- GAS was used as coated antigen to bind with the mcAb. Results： Monoclonal antibodies against Fba protein were obtained, and one of the monoclonal antibodies could especially bind Fba＋ GAS but not Fba^- GAS. The recognized epitope of the mAb might harbor in 104-108 aa of Fba protein, which just was the site that complement regulator factor H bound to Fba protein. Conclusion： Monoclonal antibodies against Fba protein are successfully obtained. One of the mAbs recognizes a natural epitope located on GAS, which is related to binding activity of Fba protein to complement regulator factor H. The result will benefit to further research in the pathogenic mechanism of Fba^＋ GAS.