Abstract: 【Objective】Pen a1 is the major allergen in shrimp, and the sensitization mechanism is related with epitopes. The epitope (187-202) of Pen a1 was chosen as the research material, the amino acids’ frequency of occurrence and their conservative property were analyzed, the mutants were synthesized and the IgE capacity of the mutants were analyzed. The critical amino acids of the epitope of Pen a1 were screened out in order to study the shrimp sensitization mechanism and provide a theoretical basis for desensitization. 【Method】 The amino acid composition and frequency of occurrence in Pen a1, all epitopes and the studied epitope were analyzed using MEGA5, and the amino acids with the highest frequency were chosen. And then the amino acids of tropomyosin in all the allergenic foods in SDAP were also investigated, and the high conservative amino acids were chosen. The common amino acids in the results of both methods were considered to be the potential critical amino acids. Then these amino acids were substituted by alanine, the wild-type peptide and the mutant peptides were then synthesized with solid phase synthesis method. The tested serum was prepared by immuning New Zealand rabbits with wild-type peptide. The capacity of IgE-binding between the wild-type peptide and the mutants were compared with iELISA and competitive Dot-blot methods. The mutant peptides whose capacity of IgE-binding showed a dramatic decline were selected, and the replaced amino acids of the peptides were recognized as the critical amino acids. 【Result】 The amino acids of glutamic acid (E), leucine (L), arginine (R), glutamine (Q), valine (V), serine (S), aspartic acid (D) were found with higher frequency occurrence in epitope than in Pen a1, and were considered as the active amino acids. E, V, L in epitope (187-202) were found with higher frequency occurrence, and were inferred to be the potential critical amino acids of studied epitope. The multiple sequence alignment of tropomyosin from allergenic foods in SDAP and Pen a1 with DNAMAN discovered that K, L, E, V, G existed in all the sequences, which indicated that the five amino acids were conservative. E, V and L which appeared in the results of both methods were selected to be substituted by alanine to synthesize the mutants 1, 2 and 3, respectively. The mutants were interacted with epitope sera to detect the IgE reactivity by competitive Dot-blot. QCA-OVA were the purified Pen a1, and the mutated peptides were competitors. The IgE reactivity against wild type and mutants were then compared. The wild-type and mutant 1 showed a similar evident inhibiting effect, mutants 2 and 3 showed a decrease in inhibiting effect. The mutant 1 with Glu to alanine showed a similar IgE reactivity to wild-type, which meant that Glu should not be the critical amino acid. Peptides with Leu (mutant 2) or Val (mutant 3) to alanine exchange showed a decrease in IgE-binding, and Leu and Val were deduced the critical amino acids of the studied epitope. The mutants were interacted with epitope sera by ELISA to compare the OD450 value with the wild-type as control. Mutant 1 showed a decrease in IgE-binding in tested epitope sera, its OD450 value was 1/2.1 of control. OD450 value of mutant 2 decreased to 1/2.6 of control, and mutant 3 decreased to 1/3.2 of control. These three mutants’ IgE-binding capacity decreased in different degrees. Combined with the results of competitive Dot-blot, Leu and Val were considered to be the critical amino acids of studied epitope. 【Conclusion】 The method for selection and identification of critical amino acids was established, L and V were the critical amino acids of epitope (187-202) in Pen a1. This method for screening the potential critical amino acids could also be used in identifying critical amino acids of other epitopes, and exploring the influence of amino acids on the IgE capacity of epitope, thus further studying the mechanism of desensitization, and can also be used in genetic engineering or amino acids modification to reduce the sensitization of allergen.

Key words: Pen a1 , epitope , critical amino acid , competitive Dot-blot , iELISA