Use this URL to cite or link to this record in EThOS:
Title: Construction and characterisation of Escherichia coli heat-labile toxin B-subunit fusion proteins
Author: Lipscombe, Martin John
ISNI:       0000 0001 3610 8695
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1991
Availability of Full Text:
Access from EThOS:
Access from Institution:
A plasmid vector was constructed which allowed for the in-frame insertion of peptide-encoding sequences at the 3’ terminal of the Escherichia coli (E. coli) heat-labile enterotoxin B-subunit (LT-B) structural gene. Several synthetic oligonucleotides, encoding various B and T cell epitopes from other proteins, were ligated into this vector. The sequence across the junctions of these novel plasmid constructs was determined and found to be as predicted. The chimeric fusion proteins expressed by these constructs were characterised in vitro by SDS-PAGE, Western blotting and GM1-linked ELISA. All the fusion proteins were shown to behave like native LT-B in that they were transported to the periplasmic space when expressed in E. coli. In addition they formed pentamers which dissociated into their constituent monomers upon boiling. Furthermore, the pentameric forms were found to retain G,,,-binding properties as determined by G,,,-linked ELISA. Some of these plasmids, expressing LT-B fusion proteins containing T cell epitopes, were transferred into an aromatic-dependent attenuated strain of Salmonella typhlmurium SL1344, and these strains were used to inoculate mice. A weak serum antibody response to one of these epitopes was demonstrated. However, a consistent in vitro T cell response to these epitopes could not be detected. Another of the fusion proteins, termed LT-B69, was partially purified by ion-exchange chromatography and used to inoculate mice intranasally. Mice immunised in this way developed serum antibodies against LT-B and P.69 (an important Bordetella pertussis antigen). Additionally, LT-B-specific and P.69-specific antibody secreting cells could be detected in their lungs. There was some evidence to suggest that these mice were slightly protected against colonisation by B. pertussis after an aerosol challenge with live organisms, compared to the levels of colonisation in a control group.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology