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

A completely synthetic gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, ∼50 kD) was constructed with oligonucleotides. After being 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 30 mg/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 the 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-level expressive AHc could not only be produced by the prokaryotic expression system built in the lab, but also owned strong immunogenicity to prepare an antitoxin for treatment and as a subunit candidate vaccine for prophylaxis against botulinum toxin serotype A.