抗A型肉毒毒素卵黄抗体的制备、纯化及活性检测  

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

作　　者：游哲荣[1] 马臣杰[1] 辛文文[1] 杨浩[1] 康琳[1] 高姗[1] 王景林[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071

出　　处：《军事医学》2014年第3期174-177,180,共5页Military Medical Sciences

基　　金：全军"十二五"重大专项资助项目(AWS11C001)

摘　　要：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∶100 000。小鼠体内中和实验证实,IgY可完全中和20LD50A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。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 .

关 键 词：肉毒梭菌毒素 A型 卵黄抗体 中和活性 

分 类 号：R392[医药卫生—免疫学]