Pen a1抗原表位187-202关键氨基酸的筛选和鉴定

【目的】Pen a1是虾中主要的过敏原蛋白，其抗原表位与致敏作用有关。对Pen a1的一个抗原表位187—202中氨基酸的出现频率及保守性进行分析后合成突变肽，并检测该突变肽与表位抗体的结合能力，筛选关键氨基酸，为研究虾致敏机理及脱敏方法提供理论依据。【方法】利用MEGA5软件对Pen a1蛋白5个表位及其氨基酸的组成与出现频率进行分析，选出这些表位中出现频率大的氨基酸；对过敏原数据库中所有致敏食物原肌球蛋白的氨基酸序列的保守性进行分析，筛选出保守性高的氨基酸。两种方法筛选出的共有氨基酸为潜在的关键氨基酸。用丙氨酸分别替代这些潜在的氨基酸形成突变肽。利用固相合成法分别合成原表位肽及突变肽。并将原表位肽作为免疫原免疫新西兰大白兔，获得表位多克隆抗体。利用间接ELISA方法及竞争性Dot-blot方法检测突变的表位肽与表位抗体IgE结合能力，筛选出结合能力明显下降的突变肽，其中替换的氨基酸即为该表位的关键氨基酸。【结果】谷氨酸（E）、亮氨酸（L）、精氨酸（R）、谷氨酰胺（Q）、缬氨酸（V）、丝氨酸（S）、天冬氨酸（D）在表位中出现频率较大，并高于在Pen a1整体蛋白中出现的概率，为活性氨基酸。将序列在187—202的表位中氨基酸组成及出现频率进行统计，推测E、V、L可能为该表位的关键氨基酸。将SDAP数据库中致敏食物原肌球蛋白序列与Pen a1氨基酸序列用DNAMAN软件进行多序列比对，K、L、E、V、G在所有比对的序列中都存在，表明这5个氨基酸为保守氨基酸。选择共有的E、V、L为可能的关键氨基酸，用丙氨酸替代表位中E、V、L分别合成1、2和3号突变肽。用竞争性Dot-blot检测突变肽结合抗体能力，以提取纯化的虾致敏蛋白为包被原，用突变肽抑制虾蛋白结合抗体，发现原表位肽抑制作用明显，1号突变肽的抑制作用与原表位肽相似，2、3号肽的抑制作用明显低于原表位肽。谷氨酸突变的1号突变肽对表位活性并没有较大影响，说明谷氨酸不是该表位的关键氨基酸；而2、3号突变肽与抗体的结合能力明显降低，即亮氨酸和缬氨酸是该表位的关键氨基酸。同时，利用间接ELISA方法，将原表位肽和突变肽与兔血清反应，比较OD450值。结果显示，1号突变肽致敏性降低，OD450值约为对照的1/2.1，2号突变肽OD450值降低为对照的1/2.6，3号突变肽OD450值降低为对照的1/3.2。说明突变表位与表位抗体结合能力均有不同程度的降低。结合竞争Dot-blot试验结果，亮氨酸和缬氨酸为该抗原表位的关键氨基酸。【结论】建立了一种关键氨基酸的筛选和鉴定的方法。亮氨酸和缬氨酸被丙氨酸取代后，其与表位对应抗体的结合能力发生明显下降，是Pen a1中表位187—202的关键氨基酸。这种筛选关键氨基酸的方法可以用于其它抗原表位的关键氨基酸的确定及氨基酸对表位致敏性的影响，深入研究过敏原脱敏机理；同时也可用于基因工程或氨基酸修饰中降低过敏原致敏性。

Selection and Identification of Critical Amino Acids in Epitope 187-202 of Pen a1

【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.