RECOMBINANT TOXOIDS OF BACTERIAL EXOTOXINS

产生免疫原性、无毒形式的细菌毒素（类毒素）是 重组DNA技术的一个潜在的重要应用。 的 本项目有两个主要目标：（1）产生有效的 铜绿假单胞菌外毒素A（ETA）的重组类毒素，和（2） 提供一种通过诱变活性物质产生类毒素的模型系统， 克隆毒素的位点残基。 我们用光亲和标记 确定谷氨酸-553（Glu-553，或E553）是一种重要的活性物质， ETA位点残基，并采用寡核苷酸定向突变， 删除该残基或用其他氨基酸取代它。 在继续 在这些研究中，我们将重点关注ETA-E553 X，这是一种候选类毒素， 特别是删除Glu-553。 我们将开发改进的方法， 大量生产ETA-E553 X和其它克隆的重组抗原， P.铜绿，并进行更彻底的表征， ETA-E553 X的生物学和生物化学性质。 这种变异毒素 将在人体中进行I期安全性和免疫原性试验， 并将用于制备蛋白质-多糖缀合物， 各种铜绿假单胞菌多糖组分。 我们还将产生 并表征ETA的其它候选重组类毒素。 这些 研究可能在控制铜绿假单胞菌方面具有重要应用 感染;以及通过以下方式获得的关于类毒素构建的经验教训： 诱变活性位点残基可以广泛应用。

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.