Yu Yun-Zhou, Li Na, Wang Rui-Lin, Zhu Heng-Qi, Wang Shuang, Yu Wei-Yuan, Sun Zhi-Wei
Beijing Institute of Biotechnology, 20 Dongdajie St., Fengtai District, Beijing 100071, China.
Clin Vaccine Immunol. 2008 Dec;15(12):1819-23. doi: 10.1128/CVI.00239-08. Epub 2008 Oct 8.
A new gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype F (FHc) was designed and completely synthesized with oligonucleotides. A soluble recombinant Hc of C. botulinum neurotoxin serotype F was highly expressed in Escherichia coli with this synthetic FHc gene. Subsequently, the purified FHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype F (BoNT/F). After the administration of FHc protein mixed with Freund adjuvant via the subcutaneous route, a strong protective immune response was elicited in the vaccinated mice. Mice that were given two or three vaccinations with a dosage of 1 or 10 microg of FHc were completely protected against an intraperitoneal administration of 20,000 50% lethal doses (LD50) of BoNT/F. The BoNT/F neutralization assay showed that the sera from these vaccinated mice contained high titers of protective antibodies. Furthermore, mice were vaccinated once, twice, or three times at four different dosages of FHc using Alhydrogel (Sigma) adjuvant via the intramuscular route and subsequently challenged with 20,000 LD50 of neurotoxin serotype F. A dose response was observed in both the antibody titer and the protective efficacy with increasing dosage of FHc and number of vaccinations. Mice that received one injection of 5 microg or two injections of >or=0.04 microg of FHc were completely protected. These findings suggest that the recombinant FHc expressed in E. coli is efficacious in protecting mice against challenge with BoNT/F and that the recombinant FHc subunit vaccine may be useful in humans.
设计了一种编码肉毒杆菌神经毒素F型（BoNT/F）Hc结构域的新基因（FHc），并使用寡核苷酸将其完全合成。利用该合成的FHc基因在大肠杆菌中高效表达了一种可溶性重组肉毒杆菌神经毒素F型Hc。随后，用纯化的FHc对小鼠进行免疫接种，并评估它们在受到活性肉毒杆菌神经毒素F型（BoNT/F）攻击后的存活情况。通过皮下途径给予与弗氏佐剂混合的FHc蛋白后，在接种疫苗的小鼠中引发了强烈的保护性免疫反应。用1或10μg的FHc进行两次或三次接种的小鼠，对腹腔注射20,000个50%致死剂量（LD50）的BoNT/F具有完全的保护作用。BoNT/F中和试验表明，这些接种疫苗小鼠的血清中含有高滴度的保护性抗体。此外，通过肌肉途径使用氢氧化铝佐剂（Sigma），以四种不同剂量的FHc对小鼠进行一次、两次或三次接种，随后用20,000 LD50的神经毒素F型进行攻击。随着FHc剂量的增加和接种次数的增加，在抗体滴度和保护效力方面均观察到剂量反应。接受一次5μg注射或两次≥0.04μg FHc注射的小鼠得到了完全保护。这些发现表明，在大肠杆菌中表达的重组FHc在保护小鼠免受BoNT/F攻击方面是有效的，并且重组FHc亚单位疫苗可能对人类有用。
