Molecular cloning and expression of ptxA, the gene encoding the 120-kilodalton cytotoxin of Actinobacillus pleuropneumoniae serotype 2
Molecular cloning and expression of ptxA, the gene encoding the 120-kilodalton cytotoxin of Actinobacillus pleuropneumoniae serotype 2
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胸膜肺炎放线杆菌血清型 2 的 120 千道尔顿细胞毒素编码基因 ptxA 的分子克隆和表达
DOI：
10.1128/iai.60.7.2726-2732.1992
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发表时间：
1992
影响因子：
3.1
通讯作者：
Andrew N. RYCROFTt
中科院分区：
文献类型：
--
作者：
Julie Macdonald;Andrew N. RYCROFTt
胸膜肺炎放线杆菌血清型2的120 kDa的细胞毒素的遗传决定簇分离从λ DNA文库的空斑免疫印迹技术。120-kDa多肽的表达通过感染的大肠杆菌细胞裂解物的Western免疫印迹分析证实，所述细胞裂解物在体外对猪肺泡巨噬细胞显示出毒性。将该毒素的遗传决定簇亚克隆到质粒载体pUC 18中。该质粒（pPTX 1）在大肠杆菌中可指导活性120-kDa细胞毒素的合成和分泌。杆菌重组毒素与A.在分子大小、蛋白质印迹分析中的反应、热不稳定性、细胞毒性活性和血清抗体的中和方面，鉴定胸膜肺炎血清型2。制备pPTX 1的限制性内切酶切割图，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。用位于ptxA基因内的1.7 kb PvuII片段进行Southern杂交分析，发现血清型参考菌株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该探针识别的序列。然而，在伴放线放线杆菌和放线杆菌中检测到相关序列（大于71%的同源性）。平等。ptxA探针与携带溶血性巴氏杆菌lktAC基因的pLKT 5和携带A.胸膜肺炎这种细胞毒素的遗传决定因素的分离将使调查的结构和组织的ptx DNA区域和进一步分析其在胸膜肺炎的发病机制中的作用。
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.