抗A型肉毒毒素卵黄抗体的制备、纯化及活性检测

目的：制备特异性的抗A型肉毒毒素（ BoNT/A）的卵黄抗体（ IgY）并分析抗体的生物活性。方法人工合成密码子优化BoNT/A重链碳端蛋白（ AHc）基因片段，并构建含有该目的片段的重组表达质粒pTIG-Trx-AHc。诱导表达的AHc蛋白经纯化定量后免疫健康母鸡，4次免疫后通过水稀释和硫酸铵盐析法从鸡蛋中提取特异性IgY。在小鼠模型中测定纯化后IgY的中和能力。结果和结论 AHc蛋白在大肠杆菌中获得了可溶性表达，占菌体裂解液上清的20％。纯化后的IgY纯度较高，效价超过1∶100000。小鼠体内中和实验证实，IgY可完全中和20 LD50 A型天然肉毒毒素，在预防和治疗肉毒中毒表现出良好的应用前景。

Preparation and purification of IgY antibody against BoNT/A and its biological activity

Objective To prepare the egg yolk immunoglobulin （IgY） antibody against botulinum neurotoxin serotype A.Methods The optimized AHc gene was synthesized and cloned into prokaryotic expression plasmid pTIG -Trx and the resulting recombinant vector pTIG-Trx-AHc was transformed into Escherichia coli BL21（DE3） strain.AHc,purified by Ni-column affinity chromatography method , was used as an immunogen for IgY production .Anti-AHc IgY was purified by wa-ter dilution method under acidic conditions and identified by SDS-PAGE.The antibody titers of IgY were evaluated by ELISA and neutralization assay in mouse models .Results and Conclusion The recombinant AHc protein was expressed in E.coli BL21（DE3） in a soluble form which amounted to 20%of the total protein.Titers of IgY increased gradually with quartic boosting vaccination and finally reached a level of 1∶100 000 .In vivo, IgY could protect mice from death caused by injection of toxin at a 20 LD50 dose.The results suggest that anti-BHc IgY antibody can be considered a preventive and ther-apeutic intervention for botulism .