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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 a specific yolk antibody (IgY) against botulinum toxin type A (BoNT / A) and analyze the biological activity of the antibody. Methods Synthetic codon-optimized BoNT / A heavy chain C-terminal protein (AHc) gene fragment was constructed and a recombinant plasmid pTIG-Trx-AHc containing the target fragment was constructed. The induced expression of AHc protein was purified after immunization of healthy hens, 4 immunizations after dilution by water and ammonium sulfate salting extract specific IgY from eggs. The neutralizing capacity of purified IgY was determined in a mouse model. RESULTS AND CONCLUSION AHc protein was expressed soluble in E. coli, accounting for 20% of the bacterial lysate supernatant. Purified IgY higher purity, titer more than 1: 000 000. In vivo neutralization experiments in mice confirmed that, IgY can completely neutralize 20LD50A natural botulinum toxin in the prevention and treatment of botulism shows good prospects.