Cloning and expression in Escherichia coli of three Rickettsia prowazekii genes, coding outer membrane proteins

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 recomb...

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Bibliographic Details
Published inMolekuliarnaia biologiia Vol. 26; no. 4; p. 847
Main Authors Emel'ianov, V V, Balaeva, N M
Format Journal Article
LanguageRussian
Published Russia (Federation) 01.07.1992
Subjects
Antibodies, Bacterial - immunology
Bacterial Outer Membrane Proteins - genetics
Bacteriophage lambda - genetics
beta-Galactosidase - genetics
Blotting, Western
Cloning, Molecular
DNA, Bacterial
Epitopes - immunology
Escherichia coli - genetics
Gene Expression
Genes, Bacterial
Genetic Vectors
Humans
Recombinant Fusion Proteins - genetics
Rickettsia prowazekii - genetics
Rickettsia prowazekii - immunology
Typhus, Endemic Flea-Borne - blood
Typhus, Endemic Flea-Borne - immunology
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Summary: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 synthesized the complete 60 K protein, and others synthesized beta-galactosidase fusion polypeptides containing epitopes of 134 K protein of the R. prowazekii outer membrane. The amplified genomic library was screened with monospecific antibodies directed against abundant 31 K and 29.5 K outer membrane proteins. Several recombinant clones expressing full or part of 29.5 K polypeptide, and none expressing 31 K polypeptide were revealed. The serum of a patient convalescing from epidemic typhus did not react in western blot with recombinant 29.5 K protein.
ISSN:0026-8984