抗眼镜王蛇毒鸡卵黄抗体的研制及初步应用

Anti-King cobra egg yolk antibody from the egg yolks of immunized hens and its primary application

目的 研制抗眼镜王蛇毒鸡卵黄抗体 ,并研究该抗体在鸡卵黄中表达过程。方法 以甲醛减毒处理的眼镜王蛇粗毒免疫莱航母鸡 ,母鸡免疫应答后 ,在卵黄中产生抗眼镜王蛇毒抗体 ,应用嗜硫色谱柱HiTrapIgYPurificationHP从鸡卵黄中提取抗眼镜王蛇毒抗体 ;以间接ELISA法及双向免疫扩散测定抗体的效价、特异性及免疫活性 ;采用Lowry氏法测定蛋白含量 ;采用SDS PAGE法测定相对分子质量 (Mr)及鉴定抗体纯度。结果 母鸡初次免疫第 9天即有抗体产生 ,初次免疫 6 0d后抗体效价超过 1∶10 0 0 0 0 ,卵黄抗体经嗜硫色谱柱纯化后 ,经SDS PAGE检测为电泳纯 ,纯化后抗体效价较纯化前提高 4倍 ,每枚蛋中平均可获得较纯抗体 97.5mg。动物体外中和试验表明 ,特异性IgY能有效中和眼镜王蛇毒 ,阻止眼镜王蛇毒对小白鼠的攻击。结论 研制并鉴定了抗眼镜王蛇毒鸡卵黄抗体 ,分析抗体在产蛋期卵黄液中表达的进度 ,为今后该抗体的持续、高质量诱导奠定基础 。

Objective To develop King cobra antivenom from t he egg yolks of immunized hens and to study the dynamic expression of IgY in egg yolks. Methods Chickens(white Leghorn) were immunized wi th King cobra venom detoxicated by formaldehyde. Egg yolk antibody (IgY )were is olated by thiophilic interaction chromatography and identified by SDS-PAGE. Act ivity of IgY was evaluated by enzyme-linked immunoserbent assay(ELISA) and doub le immunodiffusion. Protein was measured using folin-lowry method. R esults Specific antivenom could be detected in the eggs laid by the hens of 9 d after immunization. At the 60 th d after primary immunization, ELISA titers reached 1∶100 000, and 97.5mg IgY/ml yolk was obtained from thioph ilic interaction chromatography. Neutralization of King cobra venom in vivo by IgY is demonstrated in experimental mice. Conclusion K ing cobra IgY was obtained and purified from the egg yolks of immunized hens. [