Petersen S K, Foged N T
Genetic Engineering Group, Technical University of Denmark, Lyngby.
Infect Immun. 1989 Dec;57(12):3907-13. doi: 10.1128/iai.57.12.3907-3913.1989.
A chromosomal DNA library of a toxigenic type D strain of Pasteurella multocida subsp. multocida was established in Escherichia coli. From this library two clones, SPE308 and SPE312, were identified by using a monoclonal antibody against the osteoclast-stimulating P. multocida toxin (PMT). Extracts of these clones showed cytopathic activity identical to that of extracts of toxigenic P. multocida. The recombinant plasmids, pSPE308 and pSPE312, directed the synthesis of a protein with an apparent molecular weight of 143,000 which could be specifically detected by anti-PMT antibody. The recombinant toxin, which was located in the cytoplasm of E. coli, was purified by affinity chromatography with immobilized monoclonal antibody and was shown to react in a manner identical to that of PMT in a quantitative structural test using a series of monoclonal antibodies as well as in all quantitative functional tests used, i.e., tests for dermonecrotic activity and mouse lethality and the embryonic bovine lung cell test for cytopathic activity. The gene encoding this toxic activity was named toxA and was found to be present in the chromosome of toxigenic strains only of P. multocida. A probe spanning the toxA gene therefore has potential in the diagnosis and surveillance of progressive atrophic rhinitis in pigs.
在大肠杆菌中构建了多杀性巴氏杆菌多杀亚种产毒D型菌株的染色体DNA文库。利用抗破骨细胞刺激多杀性巴氏杆菌毒素（PMT）的单克隆抗体，从该文库中鉴定出两个克隆，即SPE308和SPE312。这些克隆的提取物显示出与产毒多杀性巴氏杆菌提取物相同的细胞病变活性。重组质粒pSPE308和pSPE312指导合成一种表观分子量为143,000的蛋白质，该蛋白质可被抗PMT抗体特异性检测到。位于大肠杆菌细胞质中的重组毒素，通过用固定化单克隆抗体进行亲和层析纯化，并在使用一系列单克隆抗体的定量结构试验以及所有使用的定量功能试验中，即皮肤坏死活性试验、小鼠致死试验和胚胎牛肺细胞病变活性试验中，显示出与PMT相同的反应方式。编码这种毒性活性的基因被命名为toxA，并且发现仅存在于多杀性巴氏杆菌产毒菌株的染色体中。因此，跨越toxA基因的探针在猪进行性萎缩性鼻炎的诊断和监测中具有潜力。
