RECOMBINANT TOXOIDS OF BACTERIAL EXOTOXINS

细菌外毒素的重组类毒素

• 批准号: 3134434
• 负责人: ROBERT JOHN COLLIER
• 金额: $ 11.85万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3134434
• 关键词: Escherichia coli; Pseudomonas aeruginosa; affinity labeling; bacterial vaccines; biotechnology; chemical structure; exotoxins; genetic manipulation; high performance liquid chromatography; immunological substance; molecular cloning; molecular site; point mutation; synthetic enzyme; synthetic vaccines; toxoids; virulence

Pseudomonas aeruginosa is an opportunistic pathogen which causes severe and often fatal infections in compromised or immunosuppressed individuals. Among the various extracellular products secreted by P. aeruginosa, exotoxin A (a potent inhibitor of eukaryotic protein synthesis) is the most toxic, and available evidence suggests that this toxin is a major virulence factor. Available evidence indicates that active or passive immunization against exotoxin A may be of value in the prophylaxis and treatment of infections with P. aeruginosa. Support is requested for the preparation of non-toxic forms (toxoids) of P. aeruginosa exotoxin A, to be used for vaccine production. The approach involves combining recombinant DNA techniques with a novel photochemical reaction recently discovered in this laboratory, and will include (i) photochemical identification of a residue (or residues) within the catalytic center of the toxin or of cloned toxin fragments; (ii) construction of enzymically inactive mutant toxins in Escherichia coli by site-directed or deletion mutagenesis of active site residues; (iii) development of conditions for production and purification of these inactive forms of exotoxin A; and (iv) biochemical and biological characterization to identify those non-toxic constructs which retain both immunogenicity and antigenicity. By selecting specific amino acid substitutions, based upon photochemical data and the known three-dimensional structure, potential reversion of non-toxic constructs, as well as alterations of secondary and tertiary structure, will be minimized. Ideally suited to the case of exotoxin A, this approach may prove generally useful in the construction of effective toxoids for other ADP-ribosylating exotoxins.

铜绿假单胞菌是一种条件致病菌，可引起严重和严重的感染。 对于受损或免疫抑制的个体来说，通常是致命的感染。 在铜绿假单胞菌分泌的各种细胞外产物中， 外毒素A（真核蛋白质合成的有效抑制剂）是最有效的 有毒，现有证据表明这种毒素是一种主要毒力 因素。 现有证据表明主动或被动免疫 抗外毒素A可能对预防和治疗以下疾病具有价值 铜绿假单胞菌感染。 请求支持制备 P. 的无毒形式（类毒素）。 铜绿假单胞菌外毒素A，用于疫苗生产。 方法 涉及将重组 DNA 技术与新型光化学技术相结合 该实验室最近发现的反应，将包括（i） 光化学鉴定残留物（或多个残留物） 毒素或克隆毒素片段的催化中心； (二) 大肠杆菌中酶失活突变毒素的构建 活性位点残基的定点突变或缺失突变； (三) 开发这些非活性物质的生产和纯化条件 外毒素A的形式； (iv) 生化和生物学特性 鉴定那些保留免疫原性和 抗原性。 通过选择特定的氨基酸取代，基于 光化学数据和已知的三维结构、电势 无毒结构的回复，以及二级和二级结构的改变 三级结构将被最小化。 非常适合以下情况 外毒素 A，这种方法可能在构建 对其他 ADP 核糖基化外毒素有效的类毒素。