Smith L A
Department of Immunology and Molecular Biology, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA.
Toxicon. 1998 Nov;36(11):1539-48. doi: 10.1016/s0041-0101(98)00146-9.
Synthetic genes encoding non-toxic, carboxyl-terminal regions (approximately 50 kDa) of botulinum neurotoxin (BoNT) serotypes A and B (referred to as fragment C or HC) were constructed and cloned into the methylotropic yeast, Pichia pastoris. Genes specifying BoNTA(HC) and BoNTB(HC) were expressed as both intracellular and secreted products. Recombinants, expressed intracellularly, yielded products with the expected molecular weight as judged by SDS PAGE and Western blot (immunoblot) analysis, while secreted products were larger due to glycosylation. Gene products were used to vaccinate mice and evaluated for their ability to elicit protective antibody titers in vivo. Mice given three intramuscular vaccinations with yeast supernatant containing glycosylated BoNTA(HC) were protected against an intraperitoneal challenge of 10(6) 50% mouse lethal doses (MLD50) of serotype A neurotoxin, a result not duplicated by its BoNTB(HC) counterpart. Vaccinating mice with cytoplasmically produced BoNTA(HC) and BoNTB(HC) protected animals from a challenge of 10(6) MLD50 of serotype A and B toxins, respectively. Because of the glycosylation encountered with secreted BoNT(HC), our efforts focused on the production and purification of products from intracellular expression.
构建了编码肉毒杆菌神经毒素（BoNT）A 型和 B 型无毒羧基末端区域（约 50 kDa）的合成基因（称为片段 C 或 HC），并将其克隆到甲基营养型酵母毕赤酵母中。指定 BoNTA(HC)和 BoNTB(HC)的基因作为细胞内产物和分泌产物进行表达。通过 SDS-PAGE 和 Western blot（免疫印迹）分析判断，细胞内表达的重组体产生了预期分子量的产物，而分泌产物由于糖基化而更大。基因产物用于给小鼠接种疫苗，并评估其在体内引发保护性抗体滴度的能力。用含有糖基化 BoNTA(HC)的酵母上清液进行三次肌肉内接种的小鼠，可免受腹腔注射 10(6) 50%小鼠致死剂量（MLD50）的 A 型神经毒素的攻击，其 BoNTB(HC)对应物未重复这一结果。用细胞内产生的 BoNTA(HC)和 BoNTB(HC)给小鼠接种疫苗，分别保护动物免受 10(6) MLD50 的 A 型和 B 型毒素的攻击。由于分泌的 BoNT(HC)存在糖基化现象，我们的工作重点是细胞内表达产物的生产和纯化。
