Carter, Penelope Jane

2019-02-15T14:34:59Z

2019-02-15T14:34:59Z

1996

The envelope glycoproteins of Maedi-Visna virus consist of a surface glycoprotein (gpl35) which is responsible for the characteristic spikes on the surface of the virion, and a transmembrane protein (gp41) whose function includes linkage to the surface glycoprotein, anchoring it to the virion envelope. The external glycoprotein is required for attachment to the host cell via a receptor molecule present on the surface of the cell. Cells of the macrophage lineage are the main target cells in MVV infection in vivo. The host humoral response is targeted to the surface glycoprotein resulting in neutralizing antibody production. The relevance of these antibodies is not understood as virus infection persists despite this active immune response. The external glycoprotein has also been shown to be susceptible to antigenic variation.

Expression of gpl35 as three overlapping fragments in the bacterial pGEX system was undertaken with a view of using the recombinant protein as a source of immunogen to raise monoclonal antibodies. These and the three recombinant fragments could be used for epitope mapping. However, these fragments proved to be toxic to bacterial cells resulting in low yields and high levels of contamination. In depth studies were carried out to improve the yield and attempts were made to raise immune polyclonal sera. Characterization of these sera is described.

Recombinant protein studies were extended to express gpl35 in the baculovirus expression system. This resulted in a reliable source of recombinant protein that was devoid of contamination and was easily purified. This protein was glycosylated and was recognised by MW-infected sheep sera. Preliminary studies were carried out to determine its interaction with sheep fibroblasts and hence its use to isolate the host cell receptor.

Attempts were made to raise monoclonal antibodies against gpl35 purified from virions by lectin affinity chromatography. The development of a screening assay is described. This approach did not result in the generation of any anti-gpl35 monoclonals. The preparation of polyclonal antisera raised against two peptides within the external glycoprotein is reported.

http://hdl.handle.net/1842/35130

The University of Edinburgh

Annexe Thesis Digitisation Project 2019 Block 22

Already catalogued

Characterization of the external envelope glycoprotein of maedi-visna virus, an ovine lentivirus

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy