Dertzbaugh M T, West M W
Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702-5011, USA.
Vaccine. 1996 Nov;14(16):1538-44. doi: 10.1016/s0264-410x(96)00094-1.
The purpose of this study was to identify the location of domains within the serotype A neurotoxin of Clostridium botulinum (BoNT/A) that conferred protection against botulism. The BoNT/A gene was subcloned into a series of 10 overlapping fragments that were expressed in Escherichia coli. The expressed proteins were partially purified and used to immunize mice. The resulting antisera were screened by immunoblotting analysis for the presence of BoNT/A-specific antibody. All fragments, except one, elicited antibody that recognized BoNT/A in an immunoblot. Serological screening identified several fragment-specific cross-reactive epitopes that were shared by heterologous serotypes of BoNT. Most of these epitopes immunoreactive by enzyme-linked immunosorbent assay, but not by immunoblot. Only two fragments were shown to confer protection against BoNT/A intoxication. Both of these proteins were derived from segments of the heavy chain and encoded amino acid residues H455-661 and H1150-1289 of BoNT/A.
本研究的目的是确定肉毒梭菌A血清型神经毒素（BoNT/A）中赋予抗肉毒中毒保护作用的结构域位置。将BoNT/A基因亚克隆到一系列10个重叠片段中，这些片段在大肠杆菌中表达。表达的蛋白质经部分纯化后用于免疫小鼠。通过免疫印迹分析筛选所得抗血清中是否存在BoNT/A特异性抗体。除一个片段外，所有片段均能诱导出在免疫印迹中识别BoNT/A的抗体。血清学筛选确定了几个片段特异性交叉反应表位，这些表位为BoNT的异源血清型所共有。这些表位中的大多数通过酶联免疫吸附测定具有免疫反应性，但通过免疫印迹则不然。只有两个片段显示出赋予抗BoNT/A中毒的保护作用。这两种蛋白质均来源于重链片段，编码BoNT/A的氨基酸残基H455 - 661和H1150 - 1289。
