RECOMBINANT TOXOIDS OF BACTERIAL EXOTOXINS

产生免疫原性的无毒形式的细菌毒素(类毒素)是 重组DNA技术潜在的重要应用。这个 当前项目有两个主要目标：(1)生成有效的 重组铜绿假单胞菌外毒素A类毒素(ETA)，和(2) 提供一种通过诱变活性物质来产生类毒素的模型系统- 克隆毒素的部位残留物。我们已经使用了光亲和标记技术 为了鉴定谷氨酸-553(Glu-553，或E553)是一种至关重要的活性物质- ETA的位点残基和应用寡核苷酸定向突变 删除这个残基或用其他氨基酸取代它。在继续 这些研究将集中在ETA-E553X，一种由 明确删除Glu-553。我们将制定改进的方法，以 年批量生产ETA-E553X等克隆重组抗原 铜绿假单胞菌，并对其进行更全面的表征 ETA-E553X的生物学和生化特性这种突变毒素 将在人体上进行第一阶段的安全性和免疫原性试验， 并将用于制备蛋白质-多糖结合物 各种铜绿假单胞菌多糖组分。我们还将生成 并对ETA的其他候选重组毒素进行了鉴定。这些 研究可能在控制铜绿假单胞菌方面有重要的应用。 感染；以及从以下方面吸取的关于类毒素构建的教训 诱变活性中心残基具有广泛的应用前景。

Generating immunogenic, nontoxic forms of bacterial toxins (toxoids) is a potentially important application of recombinant DNA technology. The current project has two major goals: (1) to generate an effective recombinant toxoid of Pseudomonas aeruginosa exotoxin A (ETA), and (2) to provide a model system for generating toxoids by mutagenizing active- site residues for cloned toxins. We have used photoaffinity labelling to identify glutamic acid-553 (Glu-553, or E553) as a crucial active- site residue of ETA and employed oligonucleotide-directed mutagenesis to delete this residue or replace it with other amino acids. In continuing these studies we will focus on ETA-E553X, a candidate toxoid formed by specifically deleting Glu-553. We will develop improved methods for producing ETA-E553X and other cloned recombinant antigens in quantity in P. aeruginosa, and perform a more thorough characterization of the biological and biochemical properties of ETA-E553X. This mutant toxin will be subjected to phase 1 safety and immunogenicity trials in humans, and will be used to prepare protein-polysaccharide conjugates with various P. aeruginosa polysaccharide components. We will also generate and characterize other candidate recombinant toxoids of ETA. These studies may have important applications in controlling P. aeruginosa infections; and lessons learned about toxoid construction by mutagenizing active-site residues could be widely applicable.