Chen Rongchang, Shi Jing, Cai Kun, Tu Wei, Hou Xiaojun, Liu Hao, Xiao Le, Wang Qin, Tang Yunming, Wang Hui
State Key Laboratory of Pathogens and Biosecurity, Institute of Microbiology and Epidemiology, Beijing 100071, China.
Protein Expr Purif. 2010 May;71(1):79-84. doi: 10.1016/j.pep.2009.11.007. Epub 2009 Nov 20.
Botulinum neurotoxin serotype A (BoNT/A) is an extremely potent bacterial protein toxin. The Hc fragment of BoNT/A (AHc) was shown to be non-toxic, antigenic, and capable of eliciting a protective immunity in animals challenged with homologous BoNT. In this study, we synthesized AHc gene by using T4 DNA ligase and PCR. The AHc was expressed at a high level in Escherichia coli successfully. Because of using the Trx co-expression strain, the expressed AHc is in a soluble and active form. The yield of the purified AHc was about 70mg/L, and its purity was up to 90% through one-step affinity chromatography. The AHc was positively identified by the antibodies raised against BoNT/A using immunological-dot-blot and Western blot assays. AHc was shown to bind with gangliosides and elicit immunity against BoNT/A, indicating that the expressed and purified AHc protein retains a functionally active conformation. Furthermore, the purified AHc has a strong immunogenicity and can be used as a potential subunit candidate vaccine for botulinum toxin serotype A.
A型肉毒杆菌神经毒素（BoNT/A）是一种极其强效的细菌蛋白毒素。BoNT/A的Hc片段（AHc）被证明无毒、具有抗原性，并且能够在受到同源BoNT攻击的动物中引发保护性免疫。在本研究中，我们使用T4 DNA连接酶和PCR合成了AHc基因。AHc在大肠杆菌中成功实现了高水平表达。由于使用了Trx共表达菌株，表达的AHc呈可溶且有活性的形式。纯化后的AHc产量约为70mg/L，通过一步亲和层析其纯度高达90%。使用针对BoNT/A产生的抗体，通过免疫斑点印迹和蛋白质免疫印迹分析对AHc进行了阳性鉴定。AHc被证明能与神经节苷脂结合并引发针对BoNT/A的免疫反应，这表明表达并纯化的AHc蛋白保留了功能活性构象。此外，纯化后的AHc具有很强的免疫原性，可作为A型肉毒杆菌毒素潜在的亚单位候选疫苗。
