海虾、大豆和腰果等三种食品过敏原组分的分析研究

发布时间：2018-06-10 08:42

本文选题：slgE + IgG　；参考：《暨南大学》2006年硕士论文

【摘要】：[目的] 建立食品过敏原的豚鼠过敏症模型，研究海虾、大豆和腰果等水提粗制蛋白浸液免疫原的免疫原性；其抗血清体外致敏正常豚鼠肥大细胞，建立过敏原体外定向释放组胺，探明过敏症发生过程中致肥大细胞脱颗粒的抗体类型；Western blot鉴定分析三种食物过敏原中主要的过敏原成分。 [方法] 以海虾、大豆和腰果等水提粗制蛋白浸液作为免疫原，以氢氧化铝为佐剂免疫豚鼠，建立豚鼠过敏模型；分离正常豚鼠腹腔肥大细胞，将其分别与正常和海虾致敏的抗血清及其分离纯化的sIgE、IgG1和IgG孵育，在体外用过敏原分别激发，HPLC组胺检测法检测肥大细胞的组胺释放率；应用三种食品的水提粗制蛋白浸液的豚鼠抗血清，Western blot分析主要的过敏原组分和它们的交叉抗原，并用DEAE交换层析分离纯化海虾过敏原。 [结果] 海虾、大豆和腰果蛋白组分等几种主要食品过敏原免疫组豚鼠，取致敏豚鼠血清，用ELISA方法检测其sIgE水平，发现致敏后第14d豚鼠血清sIgE效价最高，分别达1：80，1：40和1：80。将正常豚鼠腹腔肥大细胞与分离纯化的海虾免疫的豚鼠全血清的sIgE、IgG1及IgG分别结合(致敏)，，在体外用相应过敏原激发，检测组胺定向释放率，发现IgE和IgG均参与了豚鼠过敏激发，组胺定向释放率分别为40.05±1.98％和71.43±3.47％；进一步研究发现这种作用是由免疫血清中IgG1亚类介导的，而其他IgG亚类未参与。 Western blot鉴定海虾主要过敏组分的分子量为21、36、38、40kD，其中38kD蛋白与牡蛎浸液呈交叉反应；腰果主要过敏原组分为55kD、28kD和14kD，其中14和28kD与大豆蛋白浸液呈交叉发应：大豆主要过敏组分为14、28、30、60kD。 [结论] 用海虾、大豆和腰果等水提粗制蛋白浸液作为免疫原，建立了食物豚鼠过敏模型，采用HPLC组胺检测法检测过敏原体外定向释放的组胺，发现sIgE和IgG1均可参与体外致敏豚鼠肥大细胞的组胺释放。Western blot鉴定三种食物中的过敏原组分，初步确定它们的主要过敏原组分，并发现在海虾与牡蛎，腰果与大豆存在交叉过敏原。体外致敏肥大细胞组胺释放率可作为过敏原鉴定与评价的技术。
[Abstract]:[objective] to establish an allergic guinea pig model of food allergen, to study the immunogenicity of crude protein extracts from sea shrimp, soybean and cashew nuts, and to sensitize normal guinea pig mast cells by antiserum in vitro. To establish the directional release of histamine from allergens in vitro, and to identify the type of antibodies to degranulate mast cells during anaphylactogenesis. Western blot was used to identify and analyze the main allergens in three kinds of food allergens. [methods] prawns were used. A guinea pig allergic model was established by immunizing guinea pigs with aluminum hydroxide as adjuvant and crude protein extracts from soybean and cashew nuts were used as immunogen, and normal guinea pig peritoneal mast cells were isolated. It was incubated with antiserum sensitized by normal and shrimp and purified IgG1 and IgG respectively. Histamine release rate of mast cells was detected by HPLC histamine assay by allergen stimulation in vitro. The main allergen components and their cross antigens were analyzed by Western blot with the guinea pig antiserum extracted from water extract crude protein extract of three kinds of food, and the allergen of sea shrimp was isolated and purified by DEAE exchange chromatography. [results] the sea shrimp was isolated and purified by DEAE exchange chromatography. The sensitized guinea pigs were immunized with soybean and cashew protein components. The serum of sensitized guinea pigs was detected by Elisa. It was found that the titer of Siga E was the highest in guinea pigs on the 14th day after sensitization. The normal guinea pig peritoneal mast cells were combined with Siga IgG1 and IgG in the whole serum of guinea pigs immunized with purified sea shrimp, respectively. The specific release rate of histamine was detected by using the corresponding allergens in vitro. It was found that both IgE and IgG were involved in the allergic stimulation of guinea pigs, and the specific release rates of histamine were 40.05 卤1.98% and 71.43 卤3.47, respectively. The molecular weight of the main allergy components of shrimp identified by Western blot was 21 ~ 366 ~ 38 ~ 40kD, in which 38 KD protein was cross-reacted with oyster extract. The main allergen components of cashew nuts were 55 kD, 28 KD and 14 KD, among which 14 and 28 KD were cross-distributed with soybean protein extract. The main allergy components of soybean were 14 ~ 2830 ~ 60 KD. [conclusion] crude protein extracts from sea shrimp, soybean and cashew nut were used as immunogen. A food guinea pig allergic model was established. Histamine released by allergen in vitro was detected by HPLC. It was found that Siga E and IgG1 were both involved in histamine release from mast cells of guinea pig sensitized in vitro. Western blot was used to identify the allergen components in three foods. Their main allergens were identified and cross allergens were found in shrimp and oysters cashew nuts and soybean. Histamine release rate of mast cells sensitized in vitro can be used as a technique for the identification and evaluation of allergens.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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