河虾主要过敏原单克隆抗体的制备及河虾过敏原不同组分共同表位分析

发布时间：2018-04-20 06:22

本文选题：河虾过敏原组分 + 单克隆抗体　；参考：《暨南大学》2010年硕士论文

【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。
[Abstract]:[objective] to identify the allergen components of prawn, to isolate and purify the main allergens and to analyze their sensitivities; to prepare monoclonal antibodies against main allergens of prawns; to analyze the different allergens of prawns and prawns and prawns. The cross reaction and common epitopes among the allergens in crabs are expected to provide a basis for the clinical diagnosis of shrimp allergen and the design of shrimp allergen vaccine. [methods] the crude extract of prawn was prepared from phosphate buffer solution and purified by ammonium sulfate precipitation G-50 gel chromatography and anion exchange chromatography, and the monoclonal antibody against myosin antigen was prepared by hybridoma technique. Indirect Elisa, indirect competitive ELISA was used to determine the titer and affinity of monoclonal antibodies against ascites, and indirect Elisa, competitive ELISA and Western Blot were used to analyze the cross reaction and common epitopes among different allergens of prawn, prawn and crab. [results] three species of shrimp allergens, 21kD36kD and 80kD, were isolated and purified, with the purity of 97.55.2% and 85.3%, respectively, and the reaction rate with serum IgE was 43.37.776.776.76.7. Using hybridoma technique, 8 monoclonal antibodies, such as 1D11C4C4C4C4A4A4A4A3A3A5B114A5C10A5D5 and so on, were obtained. The titer of 1D113C4C4E33 strain McAbs was 1: 64000: 1: 128000: 1 6400.1D113C4 and 4E33 strain McAb could inhibit the binding of the sera of shrimp allergens to shrimp allergens (1D112C4C4A5B11 and 4A5D54 strains), and indirect ELISA inhibition showed that, There was a significant reciprocal inhibition reaction between the allergen components of shrimp and crab. [conclusion] there are at least 9 kinds of allergens and 3 protein components, including 21kD ~ 36kD ~ (80kD), which are the main allergen components of prawns, among which 36kD allergen is the most sensitive and sensitive component. Eight 36kD specific monoclonal antibodies were obtained. There was a common epitope between 21kD 36kD allergen and 80kD allergen among the allergen components of 21 KD, and the cross reaction was found among the allergens of prawn, prawn and crab. The promyosin of 36kD has a common epitope with that of prawn and the protein component of 26kD / 36kD and 70kD of crab.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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