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Volume 139, Issue 8

Cloning, nucleotide sequence and expression in of a gene () encoding a 25 kDa major outer-membrane protein (MOMP) of Free

Abstract

SUMMARY: A genomic library derived from a virulent isolate of was constructed in JM 83 using the cloning vector pUC19. The clones were screened by filter immunoassay using rabbit polyclonal antisera and in the absence of in situ bacterial lysis one such clone, LP 116, expressed -specific antigens on the surface of Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp. Southern hybridization confirmed that the fragment was DNA. 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 the parent, the DNA-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 and but not in JM 83. This was in agreement with the molecular mass of the deduced peptide of the mature protein. Immunoblots using -specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was a polypeptide. 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 designated Furthermore, using the fertile chicken egg virulence assay, clone LP 116 producing the 25 kDa MOMP of showed an increase in virulence when compared to the parent strain.

  • Received:
  • Accepted:
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  • Published Online:
� Society for General Microbiology, 1993
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1993-08-01
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Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of Legionella pneumophila
Microbiology 139, 1715 (1993); https://doi.org/10.1099/00221287-139-8-1715
/content/journal/micro/10.1099/00221287-139-8-1715
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