鲍曼不动杆菌外膜蛋白OMP33-36抗原多表位的构建与筛选

Construction and screening of antigen epltopes on Acinetobacter baumannii outer membrane protein 33×103-36×103（0MP33-36 ）

目的筛选鲍曼不动杆菌外膜蛋白（outermembraneprotein33×103～36×103，OMP33-36）抗原的B细胞、T细胞表位。方法运用生物信息学软件预测OMP33-36蛋白分子小鼠B细胞及T细胞表位并人工合成相应肽段；构建重组质粒pET-30a—OMP33-36并原核表达、纯化获得OMP。。蛋白；以OMP33-36蛋白免疫BALB／c小鼠，5周后收集小鼠血清并分离小鼠脾脏细胞。间接ELISA法检测小鼠血清中B细胞表位抗体水平，双夹心ELISA法检测经候选T细胞表位体外刺激后脾细胞分泌IFN-γ量。结果构建候选B细胞和T细胞表位各3条，分别为PBl、PB2、PB3，PT1、FF2、PT3；间接ELISA结果显示，PB1及PB2与OMP33-36蛋白免疫组小鼠血清发生反应，其A450值与对照组相比显著升高（P〈0．05）；双夹心ELISA结果显示，PT3刺激免疫组小鼠脾脏细胞产生的IFN．-γ量与对照组相比显著升高（P〈0．05）。结论成功构建并筛选出鲍曼不动杆菌OMP33-36蛋白的2个B细胞表位PBl及PB2，1个T细胞表位PT3。

Objective To screen B and T cell antigen epitopes on Acinetobacter baumannii outer membrane protein 33×103-36×103（OMP33-36）. Methods B and T cell epitopes on OMP33-36 of Acinetobacter baumannii were predicted by bioinformatics methods and synthesized. Recombinant expression plasmid pET- 30a-OMP33-36 was cloned and used to express OMP33-36 in a prokaryotic expression system. The expressed OMP33-36 was used to immunize BALB/e mice after purification. Serum sample was collected from each mouse in immunization and negative control groups, and then analyzed by indirect ELISA with synthesized peptides to identify B cell epitopes. Splenocytes were separated from every mouse and then cultured with each of the synthesized peptides, respectively. Double sandwich ELISA was performed to detect IFN-γ secretion in the supernatant of cell cultures for screening of T cell epitopes. Results Candidates of B and T cell epitopes were constructed, which were PB1, PB2, PB3, PT1, FV2 and PT3. Results of the indirect ELISA showed that peptides PB1 and PB2 reacted with the serum samples collected from immunized mice and A450 values of the immunization group were significant higher than those of the negative control group. Compared with the negative control group, enhanced secretion of IFN-γ following peptide VI3 stimulation was observed in the immunization group as indicated by the double sandwich ELISA. Conclusion Two B cell epitopes PB1 and PB2, and one T cell epitope PT3 on the OMP33-36 ofAcinetobacter baumannii were successfully constructed and screened out.