虾过敏原Pen a1抗原表位的关键氨基酸分析

摘要

建立了一种利用表位抗体筛选虾过敏原Pen a1抗原表位中关键氨基酸的方法。利用生物信息学软件MEGA5计算Pen a1中表位的氨基酸组成和出现频率,并采用DNAMAN软件对SDAP数据库中致敏食物原肌球蛋白的氨基酸保守性分析,综合两种方法筛选出各表位可能的关键氨基酸,Epitope 1：K, E, N；Epitope 2：K, L, E；Epitope 3：E, R, D, L, Q；Epitope 5：K, L, Q。用丙氨酸取代并合成突变多肽。利用表位抗体,通过竞争性免疫斑点法及间接ELISA方法检测突变多肽的抗体结合能力,筛选出各表位的关键氨基酸。分别为：Epitope1：谷氨酰胺；Epitope2：亮氨酸和谷氨酸；Epitope3：亮氨酸和天冬氨酸；Epitope5：亮氨酸。实验结果证明了此种筛选关键氨基酸的方法的可行性,为Pen a1致敏机理的研究及利用基因修饰脱敏提供了一定的理论依据。%AscreeningmethodforcriticalaminoacidsinEpitopesusinganti-Epitopeantibodywasdeveloped. The amino acids' frequency of occurrence in Epitopes of shrimp antigen Pen a1 was calculated using MEGA5 software and their conservative property of tropomyosin in SDAP database bank was analyzed using DNAMAN software. Potential critical amino acids based on these two methods were identified, namely Epitope 1 ( K, E, N) , Epitope 2 ( K, L, E) , Epitope 3 ( E, R, D, L, Q) , Epitope 5 ( K, L, Q) . The mutated polypeptides corresponding to these Epitopes in which these critical amino acids were substituted with alanine were synthesized. The IgG binding ability of these mutants was analyzed by competitive immunodot-blot method and indirect ELISA using Epitope antibody to screen the critical amino acids. Experimental results showed that the critical amino acids of the four Epitopes were as follows: Epitope 1 ( Gln ) , Epitope 2 ( Leu and Glu ) , Epitope 3 ( Leu and Asp) , Epitope 5 ( Leu) . The feasibility of this screening method was proved and it also offered a theoretical foundation for research on sensitization mechanism of Pen a1 and desensitization using gene modification.