RECOMBINANT TOXOIDS OF BACTERIAL EXOTOXINS

铜绿假单胞菌是一种机会致病菌， 在免疫力低下或免疫抑制的个体中经常是致命的感染。 在铜绿假单胞菌分泌的各种胞外产物中， 外毒素A（一种有效的真核蛋白质合成抑制剂）是最 有毒，现有证据表明，这种毒素是一种主要的毒性， 因子 现有证据表明，主动或被动免疫 抗外毒素A的抗体可能在预防和治疗 感染铜绿假单胞菌。 要求支持制备无毒形式（类毒素）的P。 铜绿假单胞菌外毒素A，用于疫苗生产。 的方法 涉及将重组DNA技术与新型光化学技术相结合 反应最近发现在这个实验室，并将包括（i） 光化学鉴定残留物（或残留物） 毒素或克隆毒素片段的催化中心;（ii） 在大肠杆菌中构建无酶活性的突变毒素， 活性位点残基的定点诱变或缺失诱变;（iii） 开发生产和纯化这些非活性化合物的条件 外毒素A的形式;以及（iv）生物化学和生物学表征 为了鉴定那些既保留免疫原性又 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，潜力 无毒结构的逆转，以及次级和 三级结构，将被最小化。 非常适合于 外毒素A，这种方法可以证明在构建 对其他ADP-核糖基化外毒素有效的类毒素。

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.