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目的系统筛选鉴定金黄色葡萄球菌Isd A的B细胞免疫显性表位,并评价其免疫保护效果。方法采用氨基酸步移策略,合成18 mer氨基酸重叠肽段。ELISA系统筛选鉴定Isd A的B细胞免疫显性表位肽段。免疫显性表位肽与KLH偶联后免疫Balb/c小鼠。分离免疫小鼠血清,ELISA检测免疫显性表位肽诱导的Ig G抗体水平,并测定表位肽诱导产生抗体所介导的调理吞噬作用。末次免疫后2周,经尾静脉感染S.aureus Newman,监测感染攻毒后各组的小鼠的生存率以及检测主要靶器官肾脏的细菌定植量。结果 Isd A49-66aa、Isd A91-108aa、Isd A217-234aa、Isd A259-276aa均能与Isd A抗血清产生强烈Ig G抗体反应。抗Isd A49-66aa、抗Isd A91-108aa、抗Isd A217-234aa和抗Isd A259-276aa血清均能与重组的Isd A产生较强的抗原抗体反应。与Alum组相比,免疫Isd A49-66aa、Isd A91-108aa、Isd A217-234aa、Isd A259-276aa后血清中的抗体能够显著增强抗体介导的中性粒细胞的调理吞噬作用(P<0.05),同时均显著提高了小鼠的生存率,并在感染后的第3天均显著降低肾脏的细菌定植(P<0.05)。结论成功鉴定出Isd A49-66aa、Isd A91-108aa、Isd A217-234aa、Isd A259-276aa为Isd A的B细胞免疫显性表位,显性表位肽Isd A49-66aa、Isd A91-108aa、Isd A217-234aa、Isd A259-276aa具有良好的免疫原性和免疫保护作用,可以作为金葡菌疫苗研究的重要候选表位疫苗组分。
Objective To screen and identify the B cell immunodominant epitopes of Staphylococcus aureus Isd A and evaluate its immunoprotective effect. Methods Amino acid step-by-step strategy was used to synthesize 18-mer amino acid overlapping peptides. ELISA system to identify Isd A B cell immunodominant epitope peptide. Balb / c mice were immunized after conjugation of immunodominant epitope peptides to KLH. Immunized mouse sera were isolated, ELISA immunostaining epitope peptide-induced Ig G antibody levels, and measured epitope peptide-induced antibody-mediated opsonophagocytosis. Two weeks after the last immunization, S. aureus Newman was infected via the caudal vein to monitor the survival rate of mice in each group after infection challenge and to detect the bacterial colonization of the kidney of the main target organ. RESULTS Isd A49-66aa, Isd A91-108aa, Isd A217-234aa, Isd A259-276aa all reacted strongly with Isd A antisera. Anti-Isd A49-66aa, anti-Isd A91-108aa, anti-Isd A217-234aa and anti-Isd A259-276aa sera all produced strong antigen-antibody reaction with recombinant Isd A. Antibodies in sera immunized with antibodies against Isd A49-66aa, Isd A91-108aa, Isd A217-234aa, and Isd A259-276aa significantly enhanced antibody-mediated neutrophil opsonophagocytosis (P <0.05) compared with Alum group ) Significantly increased the survival rate of mice and significantly reduced the colonization of the kidneys on the third day after infection (P <0.05). Conclusion The B cell immunodominant epitopes of Isd A49-66 aa, Isd A91-108 aa, Isd A17-234 aa, Isd A259-276 aa and Isd A259-276 aa were successfully identified. The dominant epitope peptides Isd A49-66 aa, Isd A91-108 aa, Isd A217-234aa, Isd A259-276aa has good immunogenicity and immunoprotection, which can be used as an important candidate epitope vaccine component in the research of S. aureus vaccine.