凡纳滨对虾主要过敏原-原肌球蛋白单克隆抗体的制备及其过敏原表位分析  

Preparation of monoclonal antibodies against the major allergen of Litopenaeus vannamei and analysis of its allergenic epitopes

作　　者：黄建芳[1] 王彩霞[1] 向军俭[1] 吕思[1] 黄晟[1] 

机构地区：[1]暨南大学抗体工程研究中心 广东省分子免疫与抗体工程重点实验室,广州510632

出　　处：《免疫学杂志》2012年第9期746-749,共4页Immunological Journal

基　　金：国家自然科学基金(30640026)

摘　　要：目的制备凡纳滨对虾主要过敏原-原肌球蛋白的单克隆抗体(mAb),运用它们和虾过敏患者血清分析凡纳滨对虾原肌球蛋白的过敏原表位。方法纯化的凡纳滨对虾原肌球蛋白免疫Balb/c小鼠,间接ELISA和Western blot筛选并建立稳定分泌抗原肌球蛋白抗体的杂交瘤细胞株;腹水型mAbs经硫酸铵沉淀、Protein G亲和层析纯化;叠加ELISA分析mAbs的抗原结合表位;虾过敏患者血清lgE与mAbs的抑制Western blot和间接竞争ELISA分析原肌球蛋白的过敏原表位。结果共筛选出5株mAbs,它们之间叠加ELISA的叠加值均高于40%;其中B5和A5能显著抑制虾过敏患者血清IgE与原肌球蛋白的结合,抑制率分别为58.1%和48.6%,同时也能抑制66.7%和44.3%的血清IgE与虾蛋白粗提液反应。结论成功制备了5株分别结合原肌球蛋白不同抗原表位的单克隆抗体,其中B5和A5能结合其过敏原表位。Shrimp is a frequent cause of severe allergic reactions, and tropomyosin was the only major shrimp allergen identified. In this report, monoclonal antibodies （mAb） against tropomyosin of Litopenaeus vannamei were prepared to analyze allergenic epitopes of tropomyosin. Six-week-old female Balb/c mice were immunized with purified tropomyosin. Then hybridoma technique, limiting dilution, indirect ELISA, and Western blot were used to obtain positive hybridoma cell lines. Monoclonal antibodies were purified by ammonium sulfate precipitation and affinity chromatograph of Protein G. Superposition ELISA demonstrated that superposition values of the five mAbs all over 40%; Western blot showed that mAb B5 and A5 could specifically inhibited the binding of sera IgE from shrimp allergic patients to tropomyosin, and the inhibition rates were 58.1% and 48.6%, respectively. The result of indirect competitive ELISA revealed mAb B5 and A5 individually inhibited the reaction between shrimp extract and sera IgE of 66.7% and 43.3% shrimp allergic patients. In conclusion, we have obtained five anti-tropomyosin positive monoclonal antibodies with different antigenic recognition sites, and two of them recognized allergenic epitopes of tropomyosin.

关 键 词：凡纳滨对虾 主要过敏原 原肌球蛋白 单克隆抗体 过敏原表位 

分 类 号：R392.12[医药卫生—免疫学] R392.11[医药卫生—基础医学]