PDF(Pubmed)
Abstract：
Botulism is a fatal neurologic disease caused by the botulinum toxin (BoNT) produced by Clostridium botulinum. It is a rare but highly toxic disease with symptoms, such as cramps, nausea, vomiting, diarrhea, dysphagia, respiratory failure, muscle weakness, and even death. Currently, two types of antitoxin are used: equine-derived heptavalent antitoxin and human-derived immunoglobulin (BabyBIG®). However, heptavalent treatment may result in hypersensitivity, whereas BabyBIG®, has a low yield. The present study focused on the development of three anti-BoNT monoclonal antibodies (mAbs), 1B18, C25, and M2, in Nicotiana benthamiana. The plant-expressed mAbs were purified and examined for size, purity and integrity by SDS-PAGE, western blotting and size-exclusion chromatography. Analysis showed that plant-produced anti-BoNT mAbs can fully assemble in plants, can be purified in a single purification step, and mostly remain as monomeric proteins. The efficiency of anti-BoNT mAbs binding to BoNT/A and B was then tested. Plant-produced 1B18 retained its ability to recognize both mBoNT/A1 and ciBoNT/B1. At the same time, the binding specificities of two other mAbs were determined: C25 for mBoNT/A1 and M2 for ciBoNT/B1. In conclusion, our results confirm the use of plants as an alternative platform for the production of anti-BoNT mAbs. This plant-based technology will serve as a versatile system for the development botulism immunotherapeutics.
摘要：
肉毒中毒是由肉毒梭菌产生的肉毒杆菌毒素（BoNT）引起的致命神经系统疾病。这是一种罕见但剧毒的疾病，有症状，比如抽筋,恶心,呕吐,腹泻,吞咽困难,呼吸衰竭,肌肉无力,甚至死亡。目前,使用两种类型的抗毒素：马源七价抗毒素和人源免疫球蛋白（BabyBIG®）。然而,七价治疗可能导致超敏反应，而BabyBIG®,产量低。本研究集中于三种抗BoNT单克隆抗体（mAb）的开发，Nicotianabenthamiana中的1B18，C25和M2。纯化植物表达的单克隆抗体，并检查大小，SDS-PAGE的纯度和完整性，蛋白质印迹和大小排阻层析。分析表明，植物产生的抗BoNT单克隆抗体可以在植物中充分组装，可以在单个纯化步骤中纯化，并且大部分保留为单体蛋白质。然后测试抗BoNTmAb与BoNT/A和B结合的效率。植物产生的1B18保留了其识别mBoNT/A1和ciBoNT/B1的能力。同时,测定另外两种mAb的结合特异性:C25用于mBoNT/A1,M2用于ciBoNT/B1。总之,我们的结果证实了使用植物作为生产抗BoNT单克隆抗体的替代平台。这种基于植物的技术将作为开发肉毒杆菌免疫治疗的通用系统。
