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首页> 外文期刊>Infection and immunity >Molecular cloning and expression of ptxA, the gene encoding the 120-kilodalton cytotoxin of Actinobacillus pleuropneumoniae serotype 2.
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Molecular cloning and expression of ptxA, the gene encoding the 120-kilodalton cytotoxin of Actinobacillus pleuropneumoniae serotype 2.

机译：ptxA的分子克隆和表达，ptxA是编码胸膜肺炎放线杆菌血清型2的120千达尔顿细胞毒素的基因。

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The genetic determinants of the 120-kDa cytotoxin of Actinobacillus pleuropneumoniae serotype 2 were isolated from a lambda DNA library by a plaque immunoblot technique. Expression of the 120-kDa polypeptide was confirmed by Western immunoblot analysis of infected Escherichia coli cell lysates, which were shown to be toxic for porcine alveolar macrophages in vitro. The genetic determinants of the toxin were subcloned into the plasmid vector pUC18. This plasmid (pPTX1) directed the synthesis and secretion of the active 120-kDa cytotoxin in E. coli. The recombinant toxin was indistinguishable from native cytotoxin from A. pleuropneumoniae serotype 2 with respect to molecular size, reaction in Western blot analysis, heat lability, cytotoxic activity, and neutralization by serum antibody. A restriction endonuclease cleavage map of pPTX1 was prepared, and deletion mutants were used to locate the minimal region of DNA required for production of intracellular toxin; this gene was termed ptxA. Southern hybridization analysis with a 1.7-kb PvuII fragment located within the ptxA gene revealed sequences with a high degree of homology in serotype reference strains 2, 3, 4, 6, and 8. Other reference strains did not contain sequences that were recognized by this probe. However, related sequences (greater than 71% homology) were detected in Actinobacillus actinomycetemcomitans and A. equuli. Weak hybridization was observed between the ptxA probe and pLKT5, which carries the lktAC genes of Pasteurella haemolytica, and between the ptxA probe and pAPH1, which carries the structural gene for type II hemolysin from A. pleuropneumoniae. The isolation of the genetic determinants of this cytotoxin will enable investigations of the structure and organization of the ptx DNA region and further analysis of its role in the pathogenesis of pleuropneumonia.
机译：通过噬菌斑免疫印迹技术从λDNA文库中分离出胸膜肺炎放线杆菌血清型2的120 kDa细胞毒素的遗传决定因素。通过感染的大肠杆菌细胞裂解物的Western免疫印迹分析证实了120 kDa多肽的表达，该蛋白在体外对猪肺泡巨噬细胞具有毒性。毒素的遗传决定子被亚克隆到质粒载体pUC18中。该质粒（pPTX1）指导大肠杆菌中活性120 kDa细胞毒素的合成和分泌。就分子大小，Western印迹分析中的反应，热不稳定性，细胞毒性活性和血清抗体中和作用而言，重组毒素与胸膜肺炎链球菌血清型2的天然细胞毒素没有区别。制备了pPTX1的限制性核酸内切酶切割图，并使用缺失突变体来定位产生细胞内毒素所需的最小DNA区域。该基因被称为ptxA。使用位于ptxA基因内的1.7-kb PvuII片段进行的Southern杂交分析显示，在血清型参考菌株2、3、4、6和8中具有高度同源性的序列。其他参考菌株不包含被该序列识别的序列探测。但是，在放线放线杆菌和马链曲霉中检测到相关序列（大于71％的同源性）。在携带溶血巴斯德氏菌lktAC基因的ptxA探针和pLKT5之间，以及携带胸膜肺炎链球菌II型溶血素的结构基因的pxA探针和pAPH1之间，观察到弱杂交。这种细胞毒素的遗传决定因素的分离将使得能够研究ptx DNA区域的结构和组织，并进一步分析其在胸膜肺炎发病机理中的作用。

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