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首页> 外文期刊>Infection and immunity >Cloning and expression of the Pasteurella multocida toxin gene, toxA, in Escherichia coli.
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Cloning and expression of the Pasteurella multocida toxin gene, toxA, in Escherichia coli.

机译：多杀性巴斯德氏菌毒素基因toxA的克隆和在大肠杆菌中的表达。

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摘要

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.
机译：产多杀性巴斯德氏菌亚种的D型毒株的染色体DNA文库。在大肠杆菌中建立了多杀菌科。通过使用抗破骨细胞刺激多杀青霉毒素（PMT）的单克隆抗体，从该文库中鉴定出两个克隆SPE308和SPE312。这些克隆的提取物显示出与产毒多杀性疟原虫提取物相同的细胞病变活性。重组质粒pSPE308和pSPE312指导了表观分子量为143,000的蛋白质的合成，该蛋白质可以通过抗PMT抗体特异性检测。通过固定化单克隆抗体的亲和色谱纯化位于大肠杆菌细胞质中的重组毒素，并在使用一系列单克隆抗体的定量结构测试中显示出与PMT相同的反应方式就像在所有使用的定量功能测试中一样，例如皮肤坏死活性和小鼠致死性测试以及胚胎牛肺细胞的细胞病变活性测试。编码这种毒性活性的基因被命名为toxA，被发现仅存在于多杀青霉的产毒菌株的染色体中。因此，跨越toxA基因的探针在诊断和监测猪进行性萎缩性鼻炎方面具有潜力。

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