河虾主要过敏组分的分离、纯化、鉴定及其致敏性分析

Identification,isolation,purification of allergens in shrimp and allergic analysis for the main allergens

目的:鉴定河虾中的过敏原组分,对主要过敏原组分进行纯化并分析其致敏性强弱。方法:用磷酸盐缓冲液(PBS)制备河虾蛋白粗提液,将其与11例虾过敏症患者血清IgE进行Westernblot,鉴定各种分子量的河虾过敏原组分;河虾蛋白粗提液用硫酸铵沉淀、G-50凝胶层析和阴离子交换层析等方法纯化其主要过敏原组分,再用虾过敏患者血清IgE进行Westernblot鉴定;并将纯化的相对分子质量(Mr)为21000、36000、800003种主要过敏原组分与虾过敏症患者血清IgE做间接ELISA,以分析其致敏性强弱。结果:West-ernblot结果显示,河虾蛋白粗提液出现9个阳性条带;其中3种主要过敏原组分21000、36000、80000与患者血清的反应率为36.4%,63.6%,45.5%;将这3种Mr的蛋白组分分别做间接ELISA,结果显示21000、36000、80000过敏原组分与11例虾过敏症患者的混合血清IgE结合的吸光值都显著高于河虾蛋白粗提液。结论:河虾中至少存在9个过敏原组分;21000、36000、8000等3种蛋白组分为河虾的主要过敏原组分,其中以36000过敏原组分致敏率和致敏性最强。进一步研究将探明21000、36000、80000等3种河虾过敏原组分的共同抗原表位,以期为明确食物过敏原检测、临床诊断和虾过敏原疫苗设计提供基础。

AIM：To identify the allergens in shrimp,and to isolate,purify and analyze the main allergen components.METHODS：The total shrimp proteins were extracted by PBS,the allergens were identified with 11 shrimp allergic patients’ serum IgE by Western blot.Three main shrimp allergens 21 000,36 000 and 80 000 were purified by ammonium sulfate precipitation,Sephadex G-50 chromatography and DEAE-exchange chromatography.Western blot and indirect ELISA were used to confirm and analyze allergenicity of the three main allergens.RESULTS：Western blot demonstrated that there were nine allergen components in shrimp proteins,and IgE binding to 21 000,36 000 and 80 000 shrimp allergen by 5,7,5 （36.4%,63.6%,45.5%） of 11 shrimp allergic patients＇ sera.Results of indirect ELISA showed that the binding absorbency of allergic patients’ sera IgE with the three main purified allergens were all higher than that with shrimp protein extraction.CONCLUSION：There are at least 9 allergens in shrimp;the 21 000,36 000 and 80 000 proteins are the main allergen components;and the 36 000 protein is the primary main allergen,with the highest allergenicity and sensitization rate.In further investigation,we will use monoclonal antibody technique to verify weather the 21 000,36 000,80 000 allergens have common epitope,which will lay a foundation for shrimp allergy clinical diagnosis and shrimp allergen vaccine design.