Leishmania parasites, protozoan parasites transmitted by female sand flies, cause the disease leishmaniasis. Leishmaniasis is responsible for 25,000–65,000 deaths and 0.7–1.0 million new cases per year. At present, there is no clinical vaccine available. Previous studies have indicated that recombinant gamma glutamylcysteine synthetase (γGCS) can protect against L. donovani and L. major infection in a murine model. However, these studies showed that production of full-length recombinant γGCS was problematic and only truncated forms of the protein could be isolated. Therefore, in this study new plasmid constructs were produced for expression of recombinant γGCS from L. donovani, L. major and L. mexicana. Molecular techniques were used to produce a series of plasmids that code for the full-length sequence of γGCS. Constructs were engineered to encode for an N-terminal T7-tag and a C-terminal His-tag, to facilitate isolation of pure and full-length γGCS protein from an Escherichia coli expression vector (pET21a(+)). Optimal expression occurred in transfected E. coli when bacteria were cultured at 18ºC after induction using 0.1 mM isopropyl β-D-1- thiogalactopyranoside. The recombinant protein was found to be present in the soluble fraction and a HisPur™ Ni-NTA spin column purification kit was used to obtain recombinant γGCS. Gel electrophoresis and western blot analysis showed that full length γGCS protein was isolated for all three recombinant proteins. However, subsequent isolation using a T7•Tag® affinity purification kit resulted in insufficient amounts of protein. Vaccine studies were completed using L. tarentolae promastigotes expressing γGCS. Mice were immunised on day 0 and 21 with parasites expressing γGCS from L. donovani alone or a mixture of parasites expressing γGCS from L. donovani, L. major or L. mexicana (triple vaccine). On day 42 mice were infected with L. donovani amastigotes and parasite burdens were determined 14 days later. Protection was associated with a mixed Th1/Th2 response based on specific IgG1 and IgG2a antibodies and on cytokines produced by antigen stimulated splenocytes used in in vitro proliferation assays.
| Date of Award | 19 Sept 2024 |
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| Original language | English |
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| Awarding Institution | - University Of Strathclyde
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| Sponsors | University of Strathclyde |
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| Supervisor | Martin Wiese (Supervisor) & Craig Roberts (Supervisor) |
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