CAJ | 학술논문

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。
목적：제비특이성적항A형육독독소（ BoNT/A）적란황항체（ IgY）병분석항체적생물활성。방법인공합성밀마자우화BoNT/A중련탄단단백（ AHc）기인편단，병구건함유해목적편단적중조표체질립pTIG-Trx-AHc。유도표체적AHc단백경순화정량후면역건강모계，4차면역후통과수희석화류산안염석법종계단중제취특이성IgY。재소서모형중측정순화후IgY적중화능력。결과화결론 AHc단백재대장간균중획득료가용성표체，점균체렬해액상청적20％。순화후적IgY순도교고，효개초과1∶100000。소서체내중화실험증실，IgY가완전중화20 LD50 A형천연육독독소，재예방화치료육독중독표현출량호적응용전경。
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 .