A型肉毒毒素Hc结构域在大肠杆菌中的高水平表达及免疫原性

High-level Expression of the Hc Domain of Clostridium botulinum Neurotoxin Serotype A in Escherichia coli and Its Immunogenicity as an Antigen

摘要:

对A型肉毒毒素受体结合区Hc基因的全序列进行优化和人工合成，获得了全长1 287bp，编码429aa的Hc基因。以pTIG-Trx为原核表达载体，实现了Hc在大肠杆菌中的高效可溶性表达及纯化，该表达水平可占可溶性全菌总蛋白的36%～53%，经一步亲和层析纯化可获得电泳级纯度的目的蛋白，在常规培养条件下，产量达到30mg/L以上。然后，纯化的重组蛋白Hc免疫小鼠后能够诱导产生高滴度特异性的抗体，也能诱导产生特异性的细胞免疫应答反应。小鼠体内A型肉毒毒素中和试验结果表明免疫组小鼠血清中含高滴度的体内中和抗体。结果表明，利用本实验的原核表达系统不仅能够高水平可溶性地表达A型肉毒毒素受体结合区Hc，而且重组Hc具有良好的免疫原性，可以用于制备治疗性抗毒素和作为亚单位候选疫苗用于预防A型肉毒毒素中毒。

Abstract:

A completely synthetic gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype A (AHc，1287bp，429aa，～50kD) was constructed with oligonucleotides. After expressed in Escherichia coli，soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%～53% of soluble total proteins) and the purified yield was more than 30mg/L by one-step purification. Then，the purified AHc was used to vaccinate Balb/c mice，which developed a strong and specific immune response as expected following administration of AHc protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble，stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab，but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.