Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of legionella pneumophila.
Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of legionella pneumophila.
复制标题
编码嗜肺军团菌 25 kDa 主要外膜蛋白 (MOMP) 的基因 (ompM) 的克隆、核苷酸序列和在大肠杆菌中的表达。
DOI：
10.1099/00221287-139-8-1715
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Rodgers,FG
中科院分区：
文献类型：
--
作者：
High,AS;Torosian,SD;Rodgers,FG
用克隆载体pUC 19在大肠杆菌JM 83中构建了嗜肺军团菌强毒株的基因组文库。用L. pneumophilarabbit多克隆抗血清，在没有原位细菌裂解的情况下，一个这样的克隆LP 116表达L. E.限制性内切酶消化分析和琼脂糖凝胶电泳显示了测量约750 bp的片段。Southern杂交证实该片段为L.嗜肺DNA测序结果表明，该片段长810 bp，开放阅读框（ORF）为678 bp。E.共亲，L.通过SDS-PAGE比较嗜肺菌DNA贡献菌株和克隆LP 116。在LP 116和L.嗜肺菌不存在E. coliJM 83.这与成熟蛋白的推测肽段的分子量一致。免疫印迹使用L.嗜肺菌特异性多克隆抗血清证实该25 kDa外膜蛋白（OMP）是嗜肺菌多肽。直接免疫荧光测定和免疫印迹使用的商业生产的单克隆抗体的主要外膜蛋白（MOMP）的共同抗原的特异性证实，由LP 116产生的25 kDa的蛋白质参与的MOMP复合物。此外，利用可育鸡蛋毒力试验，克隆LP 116产生了L.与E.共亲菌株
A genomic library derived from a virulent isolate ofLegionella pneumophilawas constructed inEscherichia coliJM 83 using the cloning vector pUC19. The clones were screened by filter immunoassay usingL. pneumophilarabbit polyclonal antisera and in the absence of in situ bacterial lysis one such clone, LP 116, expressedL. pneumophila-specific antigens on the surface ofE. coli.Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp. Southern hybridization confirmed that the fragment wasL. pneumophilaDNA. Sequencing data showed that the fragment was 810 bp in length with an open reading frame (ORF) of 678 bp. The outer-membrane profiles of theE. coliparent, theL. pneumophilaDNA-contributing strain and clone LP 116 were compared by SDS-PAGE. A protein of 25 kDa was found in outer-membrane preparations of both the clone LP 116 andL. pneumophilabut not inE. coliJM 83. This was in agreement with the molecular mass of the deduced peptide of the mature protein. Immunoblots usingL. pneumophila-specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was aL. pneumophilapolypeptide. Both direct immunofluorescence assay and immunoblots using the commercially produced monoclonal antibody specific for the common antigen of the major outer-membrane protein (MOMP) confirmed that the 25 kDa protein produced by LP 116 was involved with the MOMP complex. The gene encoding this protein has been designatedompM.Furthermore, using the fertile chicken egg virulence assay, clone LP 116 producing the 25 kDa MOMP ofL. pneumophilashowed an increase in virulence when compared to theE. coliparent strain.