A genetic approach to identify Mycobacterium bovis exported protein antigens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology, Massey University

Simple item page
dc.contributor.authorBorich, Suzanne Marie
dc.date.accessioned2011-09-29T00:07:16Z
dc.date.available2011-09-29T00:07:16Z
dc.date.issued1997
dc.description.abstractA novel approach, combining phoA-fusion technology with T cell screening of a recombinant cosmid library, was used to detect Mycobacterium bovis exported T cell antigens. An M. bovis BCG library of phoA-fusions was constructed in Escherichia coli and Mycobacterium smegmatis using the plasmid vector pJEM11. The M. bovis BCG DNA inserts from ten PhoA+ clones were partially sequenced and used to search databases for similarities to known genes. These revealed similarities to a family of genes coding for high temperature-requirement serine proteases and a Mycobacterium leprae putative exported lipoprotein gene (pel). The DNA inserts from PhoA+ clones were used to probe an M. bovis cosmid library expressed in M. smegmatis 10 identify cosmids containing the full-length genes coding for these exported proteins. Culture filtrates (CFs) prepared from selected M. smegmatis recombinants (cosmids) were assayed for their ability to induce proliferation and IFN-γ-production from peripheral blood mononuclear cells (PBMCs) taken from M. bovis BCG-immunised and non-immunised control cattle. Culture filtrates from two recombinant M. smegmatis (cosmids 44 and 56) induced significant IFN-γ-production and proliferation by PBMCs from immunised animals. An exported protein gene, identified using the phoA-fusion technology, was subcloned from cosmid 56 and its sequence determined and analysed. Database searches using the deduced amino acid sequence of this gene revealed similarities to an M. leprae putative exported lipoprotein (Pel) and a family of MalE maltose-binding proteins. The M. bovis pel gene was shown to be expressed by recombinant M. smegmatis. Preliminary evidence from this study indicates that the M. bovis Pel protein is recognised by antigen-specific lymphocytes from M. bovis BCG-immunised animals. The PBMCs taken from M. bovis challenged and M. bovis BCG vaccinated / challenged cattle also recognised CF from recombinant M. smegmatis expressing the pel gene in in vitro immunoassays. The combined strategy of using phoA-gene fusions and T cell screening of CFs from a recombinant M. bovis cosmid library proved a sensitive and rapid method for the detection of potential M. bovis T cell antigens.en_US
dc.identifier.urihttp://hdl.handle.net/10179/2733
dc.language.isoenen_US
dc.publisherMassey Universityen_US
dc.rightsThe Authoren_US
dc.subjectProteinsen_US
dc.subjectImmunologyen_US
dc.subjectAntigensen_US
dc.subjectMycobacterium bovisen_US
dc.subjectTuberculosis in cattleen_US
dc.subjectImmunological aspectsen_US
dc.subjectT cellsen_US
dc.titleA genetic approach to identify Mycobacterium bovis exported protein antigens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology, Massey Universityen_US
dc.typeThesisen_US
massey.contributor.authorBorich, Suzanne Marie
thesis.degree.disciplineMolecular Biology
thesis.degree.grantorMassey University
thesis.degree.levelDoctoral
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophy (Ph.D.)
Files
Original bundle
Now showing 1 - 2 of 2
Thumbnail Image
Name:
02_whole.pdf
Size:
3.29 MB
Format:
Adobe Portable Document Format
Description:
Download
Thumbnail Image
Name:
01_front.pdf
Size:
779.66 KB
Format:
Adobe Portable Document Format
Description:
Download
License bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
license.txt
Size:
804 B
Format:
Item-specific license agreed upon to submission
Description:
Download
Collections
Theses and Dissertations