摘要
[目的]鉴定河虾的过敏原组分,对主要过敏原进行分离纯化并分析其致敏性强弱；制备河虾主要过敏原的单克隆抗体；分析河虾不同过敏原及海虾、河虾、蟹过敏原之间的交叉反应和共同表位；以期为虾过敏症临床诊断和虾过敏原疫苗设计提供基础。
[方法]用磷酸盐缓冲液制备河虾粗提液,经硫酸铵沉淀、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；应用杂交瘤技术制备抗原肌球蛋白的单克隆抗体；间接ELISA,间接竞争ELISA测定腹水型单克隆抗体的效价和亲和力；运用间接ELISA、竞争ELISA和Western Blot等方法分析河虾不同过敏原组分及河虾、海虾、蟹过敏原之间的交叉反应和共同表位。
[结果]分离纯化获得21kD、36kD和80kD 3种河虾过敏原,纯度分别为97.5%,94.2%和85.3%,与患者血清IgE的反应率分别为43.3%,76.7%,36.7%。应用杂交瘤技术共获得1D11、2C4、3C4、4A3、4E3、4A5B11、4A5C10、4A5D5等8株单克隆抗体,其中1D11,3C4,4E33株单抗效价为1：64000,1：128000,1：6400。1D11、3C4和4E33株单抗能抑制虾过敏患者血清与虾过敏源的结合；1D11、2C4,4A5B11和4A5D54株单抗能与河虾其它的过敏原组分发生特异性反应；间接ELISA抑制结果显示,河虾、海虾和蟹的过敏原组分之间存在明显的相互抑制反应。
[结论]河虾中至少存在9种过敏原组分,21kD、36kD、80kD等3种蛋白组分为河虾的主要过敏原组分,其中以36kD过敏原组分致敏率和致敏性最强。获得8株36kD特异性单克隆抗体。河虾21kD、36kD的过敏原组分之间具有共同过敏原表位,21kD、36kD和80kD过敏原之间具有共同表位；河虾、海虾和蟹的过敏原之间具有交叉反应,河虾36kD的原肌球蛋白与海虾的原肌球蛋白及蟹的26kD、36kD和70kD的蛋白组分具有共同表位。
AIM:To identify the allergens in River shrimp, and to isolate, purify and analyze the main allergen components. Preparing the monoclonal antibody of the main shrimp allergen to analyze the shared epitope of the different allergens in river shrimp and the cross reaction between different shrimps and crabs, which will lay a foundation for shrimp allergy clinical diagnosis and shrimp allergen vaccine design.
METHOD:Three main River shrimp allergens were purified by ammonium sulfate precipitation, Sephadex G-50 chromatography and DEAE-exchange chromatography from the total river shrimp proteins. The BALB/C mice were immunized with purified 36kD allergen to produce its monoclonal antibody. Indirect ELISA, indirect competitive ELISA and Western Blot were exploited to analyze the common epitopes of the river shrimp allergens and cross reaction between river shrimp, sea shrimp and crab.
RESULTS:The purified 21kD,36kD,80kD allergens were estimated to be about 97.5%,94.2% and 85.3% pure. Western blot demonstrated that the reaction rate between main allergens 21kD, 36kD,80kD and shrimp allergic patients'sera IgE were 43.3%,76.7%,36.7%.1D11,2C4, 3C4,4A3,4E3,4A5B11,4A5C10 and 4A5D5 McAbs were obtained, and the title of 1D11,3C4 and 4E3 wasl:64000,1:128 000,1:6400. The results of indirect ELISA, competitive indirect ELISA and western blot showed:the 1D11,3C4 and 4E3 can recognized the eptiope of shrimp allergens; 1D11,2C4,4A5B1 land 4A5D5 can react with the other allegens in river shrimp. The indirect competitive ELISA demonstrated that the allergens in river shrimp, sea shrimp and crab can obviously surpress each other.
CONSULUTION:The 21kD,36kD and 80kD proteins are the important allergens in river shrimp, and the 36kD protein is the primary main allergen, with the highest allergenicity and sensitization rate. There are some common epitopes in 21kD,36kD and 80kD allergen component. Cross reaction were happened between river shrimp, sea shrimp and crab; 36kD allergen component share common epitopes with 36kD allergen component in sea shrimp and 23kD,36kD,70kD protein components in crab.
引文
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