免疫亲和层析柱纯化大豆球蛋白多克隆抗体的研究
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国家自然科学基金面上项目(31671778)


Studies on Purification of Polyclonal Antibody Against Soybean Globulin on Immunoaffinity Chromatography Column
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    摘要:

    为了得到纯度高、特异性好的抗大豆球蛋白多克隆抗体,用溴化氢活化的琼脂糖4B纯化经硫酸铵分级沉淀后的抗体,此免疫亲和层析过程节省时间且操作简单。经凝胶过滤层析的大豆球蛋白,溶胀后的填料与大豆球蛋白配基以共价键的形式连接,利用抗原抗体特异性结合的特点,杂蛋白随流动相流出,再利用洗脱液将抗大豆球蛋白的目标抗体特异性地洗脱下来,用紫外检测仪检测非特异性洗脱峰和特异性洗脱峰。通过SDS-PAGE鉴定,多抗血清中的其它蛋白组分被非特异性洗脱掉,经浓度测定,特异性洗脱的蛋白质量浓度为0.3 mg/mL,间接ELISA显示,纯化后血清稀释128倍后显示为阳性。间接竞争ELISA测定抗体特异性结果显示抗体特异性得到提高。抗体被成功纯化,为加工破坏大豆球蛋白致敏性亚分子结构定位奠定基础,从而促进低致敏性食品的研发。

    Abstract:

    In order to obtain high purity and specificity polyclonal antibody against soybean globulin, CNBr activated Sepharose-4B was used to purify the antibody which was precipitated by ammonium sulfate. The process of affinity chromatography was time-saving and simple. Purified soy globulin was covalently linked to affinity chromatography packing and the target antibody of the anti-soy globulin was specifically eluted by the nature of antigen antibody specific binding. The non-specific elution peak and the specific elution peak were obtained by affinity chromatography. Through SDS-PAGE identification, the other protein components in the multi antibody serum were washed off by a non-specific method. The concentration of the specific eluted protein was 0.3 mg/mL. The results of indirect ELISA showed that the purified serum was still positive after diluted 128 times. The purified antibody lays the foundation for destroying the structural localization of soybean globulin sensitization sub molecular, thus promoting the research and development of low sensitization food.

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姚利利;席俊;陈慧彬;陈阳;皮江一;李潘.免疫亲和层析柱纯化大豆球蛋白多克隆抗体的研究[J].中国食品学报,2021,21(6):296-300

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  • 在线发布日期: 2021-07-16
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