Molecular cloning and expression of Rickettsia prowazekii genes for three outer membrane proteins in Escherichia coli.
Rickettsia prowazekii (virulent Breinl strain) random genomic DNA fragments were cloned in the lambda gt11 expression vector by using non-palindromic adaptors. Several immunoreactive clones were selected after screening 20,000 individual recombinant plaques with human convalescent serum. Some recombinants expressed complete 60 kDa polypeptide, and others expressed beta-galactosidase fusion polypeptides containing different epitopes of 134 kDa protein of the R. prowazekii outer membrane (OM). Amplified genomic library was screened with monospecific antibodies directed against abundant 31 kDa and 29.5 kDa OM proteins (OMPs). Several recombinant clones expressing full or part of 29.5 kDa polypeptide, and none expressing 31 kDa polypeptide were revealed. The serum of a patient convalescing from epidemic typhus did not react in Western blot with recombinant 29.5 kDa protein.
Emelyanov
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Gamaleya Institute for Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow.
D000907:Antibodies, Bacterial / Q000097:blood* / Q000302:isolation & purification; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D015698:Genomic Library; D006801:Humans; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D012283:Rickettsia prowazekii / Q000235:genetics* / Q000276:immunology*; D014438:Typhus, Epidemic Louse-Borne / Q000276:immunology*
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D016056:Immunodominant Epitopes; D011993:Recombinant Fusion Proteins