The Cloning, Sequencing, Expression and Demonstration of Biological Activity of Feline Recombinant Interferon-gamm

Argyle, David John (1995) The Cloning, Sequencing, Expression and Demonstration of Biological Activity of Feline Recombinant Interferon-gamm. PhD thesis, University of Glasgow.

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Abstract

Cytokines are intercellular messengers which orchestrate host defence, repair and haemopoiesis. The potential that cytokines have in the pharmacological manipulation of the immune and haemopoeitic systems has lead them to be at the forefront of biomedical research. In human medicine, cytokines now feature in many clinical trials ranging from those involving infectious diseases to those involving the treatment of cancer. The potential benefits to therapeutics that cytokines offer is now being realised in veterinary medicine. The cat suffers from a number of neoplastic, infectious, immune mediated, and inflammatory diseases and, in addition, can provide a model for human diseases such as HIV. This thesis describes the cloning, sequencing and expression of feline recombinant interferon-gamma with the ultimate aim of exploring the potential of this cytokine in the treatment and prevention of diseases in cats. Initially feline specific cDNA, encoding interferon-gamma, was amplified using the polymerase chain reaction (PCR). Messenger RNA was harvested from feline lymphocytes which had been incubated with a non-specific mitogen. This mRNA was then used to create a library of first strand cDNA molecules, from which the interferon specific cDNA was amplified using synthetic primers and the process of PCR. Subsequently, the products of PCR were sub-cloned into plasmid vector and sequenced. The feline interferon cDNA coding region was found to be 568bp, coding for a peptide of 166 amino acids. In addition, the sequence showed a high degree of homology to the human interferon-gamma molecule. Expression of the interferon gene was achieved using the baculovirus system. This system produces a glycosylated product and can be scaled up to producing large quantities of recombinant protein. Since the ultimate aim was to use the interferon in-vivo, then both of these factors were important in the choice of expression system. Recombinant baculovirus, expressing interferon protein, was generated by homologous recombination of a transfer vector containing the interferon cDNA and linearized wild-type baculovirus. Expression and biological activity of the protein was determined by two biological assays. The first was a viral interference assay based upon the ability of interferon to protect cells against viral infection, and the second assay was based on the ability of interferon to up-regulate MHC Class II antigens on cells. Both assays confirmed that the baculovirus system was producing a biologically active protein. Immunological detection of the interferon protein was carried out using antisera raised against synthetic peptides. These peptides were selected by analysis of regions of the interferon protein sequence which were predicted to be immunogenic sites. Western blot analysis of the interferon protein, using polyclonal antisera, demonstrated the mature recombinant protein to be around 20kDa. Partial purification of the interferon protein was carried out using a combination of salt precipitation and ion exchange/FPLC chromatography. The potential benefits and drawbacks that recombinant feline interferon-gamma offers for the treatment of feline diseases is discussed in the final chapter. The advent of recombinant DNA technology, coupled with the ever increasing amount of experimental data demonstrating the biological function of these cytolcines, will undoubtedly lead to a rational approach to introducing these molecules into clinical trials. This thesis, in part, demonstrates an approach for taking a gene from the cloning stage towards clinical application.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: David Onions
Keywords: Veterinary science, Virology
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-75471
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 20:01
Last Modified: 19 Nov 2019 20:01
URI: http://theses.gla.ac.uk/id/eprint/75471

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