Expression and use of recombinant beak and feather disease virus (BFDV) capsid protein

Edward Ian Patterson

    Research output: ThesisDoctoral Thesis

    97 Downloads (Pure)

    Abstract

    The models highlight important antigenic regions, while preserving the underlying structure of the protein, providing insight to the significance of antigenic mutations when infecting new host species. To determine if, and where, BFDV Cap binds to the nuclear importin protein, importin � (Imp�), a peptide containing the nuclear localizing signal of BFDV Cap was complexed with a highly soluble isolate of Imp�, crystallized and the structure solved following X-ray crystallography. This concluded that BFDV cap is capable of binding the major site of Imp� for nuclear transport. The recombinant protein was also evaluated in a haemagglutination assay (HA), novel binding assay with susceptible host erythrocytes, and as a preliminary vaccine. The HA and binding assay were able to demonstrate mimicry of the native virus in vitro, and immunization of individuals with recombinant BFDV Cap increased their antibody titre toward BFDV. To improve consistency and convenience in diagnostic assays, a blocking ELISA (bELISA) was developed using E. coli cellular lysate containing recombinant BFDV Cap and recognition of a monoclonal antibody. Although the linear correlation and specificity were not acceptable to implement the current assay as a diagnostic assay, there is a trend toward linearity to encourage further experimentation. The results show extended use in characterization and assay development with a single isolate of BFDV Cap and a significant improvement toward large-scale expression of recombinant BFDV proteins. Additional measures should be taken to improve solubility of the protein, which would increase the likelihood of generating structural data through crystallography or nuclear magnetic resonance, maintain natural confirmation to increase robustness for binding assays to explore for facets of the viral biology, and present natural antigenic regions for more specific immunization and accurate diagnostic assays.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    • Charles Sturt University
    Supervisors/Advisors
    • Raidal, Shane, Co-Supervisor
    • Forwood, Jade, Co-Supervisor
    Award date20 Jul 2013
    Place of PublicationAustralia
    Publisher
    Publication statusPublished - 2012

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