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Intranasal immunization using the B subunit of the Escherichia coli heat‐labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen
Author(s) -
Ljpscombe M.,
Charles I. G.,
Roberts M.,
Dougan G.,
Tite J.,
Fairweather N. F.
Publication year - 1991
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1991.tb00785.x
Subject(s) - epitope , biology , bordetella pertussis , escherichia coli , microbiology and biotechnology , immunogenicity , pertussis toxin , plasmid , recombinant dna , fusion protein , oligonucleotide , diphtheria toxin , antigen , toxin , gene , biochemistry , bacteria , g protein , genetics , receptor
Summary The plasmid pBRD026, which directs expression of the B subunrt of the Escherichia coli heat‐labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3 end of the gene. An oligonucleotide linker containing restriction sites for BglH and Spel was inserted at the Spel site at the 3′ end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly–Pro–Gly–Pro which we propose acts as a ‘hinge’ between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetelia pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM 1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB‐specific and P.69‐specific antibody‐secreting cells in the lungs of immunized mice.