RECOMBINANT TOXOIDS OF BACTERIAL EXOTOXINS

细菌外毒素的重组类毒素

基本信息

批准号：
3134438
负责人：
ROBERT JOHN COLLIER
金额：
$ 17.65万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-07-01 至 1994-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3134438
关键词：
Escherichia coli Pseudomonas aeruginosa bacterial vaccines biological products biotechnology exotoxins genetic manipulation genetic recombination human subject laboratory mouse molecular cloning protein engineering protein structure function site directed mutagenesis synthetic vaccines toxoids yeasts

项目摘要

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.

产生免疫原性、无毒形式的细菌毒素（类毒素）是重组 DNA 技术的潜在重要应用。这当前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。我们使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诱变删除该残基或用其他氨基酸替换。在继续在这些研究中，我们将重点关注 ETA-E553X，这是一种候选类毒素，由特别删除 Glu-553。我们将开发改进的方法大量生产 ETA-E553X 和其他克隆重组抗原铜绿假单胞菌，并对铜绿假单胞菌进行更彻底的表征 ETA-E553X 的生物学和生化特性。这种变异毒素将在人体中进行第一阶段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成分。我们还将生成并表征了其他候选的 ETA 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。