卵黄抗体五种提取工艺比较及初步应用

Comparison and preliminary application of five extraction methods of egg yolk antibody

为了得到较优的卵黄抗体提取工艺,试验改良了辛酸法、水稀释法、氯仿法、硫酸铵沉淀法与聚乙二醇沉淀法5种提取方法。IPGT诱导表达重组猪致病性大肠杆菌(Escherichia coli)毒素STxB蛋白(rSTxB),与佐剂71VG乳化制备免疫原免疫蛋鸡,利用改良的聚乙二醇沉淀法提取高免卵黄抗体,采用SDS-PAGE电泳检测抗体纯度,通过Western blot检测卵黄抗体和血清抗体效价。结果表明,辛酸法、水稀释法、氯仿法、硫酸铵沉淀法、聚乙二醇沉淀法提取效率分别为96.00、81.99、155.50、65.34、46.00 mg/mL,其中聚乙二醇沉淀法的提取纯度最高。重组STxB蛋白分子量大小约25 kDa,血清中抗体效价最高达到64000倍,卵黄抗体最高稀释倍数为16000倍。综合评估,聚乙二醇的提取法相对较优,且提取的卵黄抗体具有良好的免疫活性。

In order to obtain a relatively good extraction process of egg yolk antibody,five methods including octanoic acid method,water dilution method,chloroform method,ammonium sulfate precipitation method and polyethylene glycol precipitation method were improved in this experiment.Expression of recombinant porcine pathogenic Escherichia coli toxin STxB(rSTxB)was induced by IPGT,and then the immunogen was prepared by emulsifying the rSTxB with the adjuvant 71VG,and the hens were immunized,IgY was ex⁃tracted by modified polyethylene glycol method.The purity of the IgY antibody was detected by SDS-PAGE electrophoresis,and the immunoactivity and titers of yolk antibody and serum antibody titer were detected by Western blot.The results showed that the extrac⁃tion efficiencies of octanoic acid method,water dilution method,chloroform method,ammonium sulfate precipitation method and poly⁃ethylene glycol precipitation method were 96.00,81.99,155.50,65.34,46.00 mg/mL,respectively,and the extraction purity of polyeth⁃ylene glycol precipitation was the highest.The molecular weight of rSTxB protein was about 25 kDa,the antibody titer in serum was up to 64000 times,and the highest dilution factor of yolk antibody was 16000 times.For the comprehensive evaluation,the extraction method of polyethylene glycol precipitation was relatively superior,and the extracted yolk antibody showed good immunological activity.