Characterization of Botulinum Toxin Subtypes - Genetics of Types

项目总结（见说明）： 约翰逊实验室对该项目的主要目标是纯化新型神经毒素的结构和功能。 机制研究，并制定对策。在过去的12个月里， 扩展了我们实验室开发的梭菌多位点序列分型（MLST）方法 肉毒杆菌包括血清型B、E和F。结果表明，相当大的系统发育多样性， 菌株谱系并鉴定新菌株。此外，多样性进行了调查，使用微阵列系统 在血清型和亚型中发现新基因。最初的阵列分析表明， 这种方法有效地描绘了多样性。我们的实验室还发现， BoNT/A3和BoNT/A4存在于约260- 280 kb的大质粒上。这是第一次展示 BoNT/A的基因存在于质粒上。C.肉毒杆菌A3和A4增加 通过改变培养基和培养条件，约10倍。新的净化方法正在研究中 BoNT/A3、BoNT/A4和与神经毒素相关的无毒蛋白质。无毒基因成分 非血凝素（NTNH）和p47蛋白已被克隆、表达并产生抗体 用于BoNT的亲和纯化。神经毒素的成功生产和纯化， 蛋白质将使蛋白质的生物化学，机械和结构研究成为可能，并提供 制定对策的材料。

PROJECT SUMMARY (See instructions): The primary goal of the Johnson lab for this project is to purify the novel neurotoxins for structural and mechanistic studies, and for development of countermeasures. During the past 12 months we have expanded the Multi Locus Sequence Typing (MLST) method developed in our laboratory for Clostridium botulinum to include serotypes B, E, and F. The results indicated considerable phylogenetic diversity of strain lineages and identified novel strains. In addition, diversity was investigated using a microarray system for the discovery of novel genes among serotypes and subtypes. Initial array analysis has shown that the diversity is effectively delineated by this approach. Our laboratory also discovered that the genes encoding BoNT/A3 and BoNT/A4 reside on large plasmids of about 260-280kb. This is the first demonstration of genes for BoNT/A being present on plasmids. Toxin production in C. botulinum A3 and A4 was increased about 10-fold by modifying the medium and culture conditions. New methods are in progress for purification of BoNT/A3, BoNT/A4 and non-toxic proteins associated with the neurotoxins. Gene components for nontoxic non-hemagglutinin (NTNH) and p47 proteins have been cloned, expressed, and antibodies produced for affinity purification of the BoNTs. The successful production and purification of the neurotoxins and nontoxic proteins will enable biochemical, mechanistic, and structural studies of the proteins, and afford materials for countermeasure development.