REGULATION OF BOTULINUM NEUROTOXIN

描述（改编自申请人的摘要）：本研究的主要目标 研究项目是揭示和分析参与调控的基因 A型肉毒梭菌菌株62 A中神经毒素（BoNT）的产生。 非毒性（tox-）突变体和BoNT显著降低的突变体 生产，以及在合成无毒成分的突变体的影响 A型毒素复合物，将在插入诱变后分离 接合转座子Tn 916 影响毒素生成的突变体 将通过免疫印迹试验和ELISA筛选菌落进行鉴定 在微量滴定板中。 详细的Southern杂交分析 将进行亲本和突变株，以定位调节基因， 梭菌染色体，并揭示哪些基因受到调控。 的 含有调控区的梭菌染色体片段将被 克隆，它们的序列确定和分析。 毒力的作用 基因将通过突变株与 含有从亲本分离的目的区域的野生型等位基因 株 BoNT和无毒的转录和翻译模式 毒素复合物的组分将在C.肉毒杆菌62 A 北方分析、蛋白质印迹和ELISA。 获得的知识 这些研究对开发改进的 肉毒杆菌毒素复合物作为药物和了解 C的能力。肉毒杆菌引起的食源性疾病和人类和动物 感染.

DESCRIPTION (Adapted from applicant's abstract): The primary goal of this research project is to reveal and analyze genes involved in the regulation of neurotoxin (BoNT) production in Clostridium botulinum type A strain 62A. Nontoxigenic (tox-) mutants and mutants with significantly decreased BoNT production, as well as mutants affected in synthesis of nontoxic components of the type A toxin complex, will be isolated after insertion mutagenesis with the conjugative transposon Tn916. Mutants affected in toxinogenesis will be identified by screening colonies by an immunoblot assay and by ELISA in microtiter plates. Detailed Southern hybridization analyses of the parent and mutant strains will be performed to locate regulatory genes on the clostridial chromosome, and to reveal which genes are regulated. The clostridial chromosomal fragments containing regulatory regions will be cloned, their sequence determined and analyzed. The role of the virulence genes will be determined by complementation of the mutant strain with the wild-type allele containing the region of interest isolated from the parent strain. Transcription and translation patterns of BoNT and nontoxic components of the toxin complex will be analyzed in C. botulinum 62A by Northern analyses, by Western blots, and by ELISA. Knowledge gained in these studies will have practical value in the development of improved botulinum toxin complexes as pharmaceuticals and for understanding the capacity of C. botulinum to cause foodborne disease and human and animal infections.