Intermediate filaments are fibrous proteins, appearing in a wide variety of tissue specific forms. The function of these proteins is poorly understood, although they are commonly believed to perform a structural role in the cell. Evidence suggests that the role these proteins play may be more dynamic than was previously believed. To gain more insight into their normal in vivo function, a single-chain monoclonal antibody has been constructed to serve as a specific reagent which can disrupt the intermediate filament network in vivo. The work presented in this thesis represents the first step in an approach which involves the use of single-chain monoclonal antibodies as specific reagents to target and disrupt the function of intracellular proteins. / The polymerase chain reaction was used for the cloning and modification of the heavy and light chain variable regions of the murine monoclonal antibody produced by the TIB 131 hybridoma. The variable regions of the light and heavy IgG chains were initially amplified from cDNA using degenerate 5$ sp prime$ primers and 3$ sp prime$ primers complementary to the constant region of the appropriate chain. The amplification products were cloned individually, sequenced, then modified to include restriction sites suitable for cloning into an expression vector. The two modified variable regions were cloned into an expression vector, and when expressed in either bacteria or in a rabbit reticulocyte lysate system, yielded a protein of the expected molecular weight.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.68253 |
| Date | January 1994 |
| Creators | Rutherford, Sharon Ann |
| Contributors | Georges, Elias (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Institute of Parasitology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001396101, proquestno: AAIMM94511, Theses scanned by UMI/ProQuest. |
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