High-level expression of the Hcc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli and its immunogenicity as an antigen.
A completely synthetic gene encoding the He domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, -50 kD) 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 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 AHe 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.
Yu
Yun-Zhou
YZ
Beijing Institute of Biotechnology, Beijing 100071, China.
Sun
Zhi-Wei
ZW
Wang
Shuang
S
Yu
Wei-Yuan
WY
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology; D019274:Botulinum Toxins, Type A / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D001906:Botulism / Q000276:immunology / Q000517:prevention & control; D047013:Clostridium botulinum type A / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005260:Female; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D019444:Vaccines, DNA; D019274:Botulinum Toxins, Type A