The main aim of this study was to develop a fast and efficient way of expressing <i>P. falciparum </i>antigenic epitopes as conformationally conserved antigenic structures whose expression can be readily ‘scaled-up’ for clinical testing. The <i>E. coli</i> and <i>P. pastoris</i> systems were used, both singly and as linked production systems using ‘shuttle vectors’ that can be dually expressed in both microorganisms. Initially a proof-of-principle experiment to express the CIDRI domain of PfEMP1 has been conducted. It was expressed and purified in good quantity and used to immunize rabbits. The sequenced 3D7 <i>P. falciparum</i> genome was then exploited to construct degenerate primers for several domains of PfEMPl (namely DBL α, β and γ). These primers were used to amplify targets from FCR3CSA parasites. PCR products were then ligated into the dual expression vector pPICHOLI1 plasmid. Several erythrocyte surface antigen expression libraries were constructed. Development and optimisation of microarray and high-throughput screening assays in malaria vaccine development was carried out to accelerate the process of identifying malaria vaccine candidates. Growth of colonies of <i>P. pastoris</i> on filters on agar plates for high-throughput screening, a novel procedure, was optimized. These libraries were screened using patients’ pooled sera from endemic areas in Sudan, as well as sera from male and pregnant women suffering from the pregnancy malaria syndrome from a holo-endemic malaria transmission zone, Ghana. Different domains from a gene of particular interest, the NF54 <i>var</i>2CSA gene were also amplified for study. The domains of DBL3X and DBL4e have been produced and purified from mid-scale induction experiments.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640381 |
| Date | January 2006 |
| Creators | Alkahtani, Saad M. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/12491 |
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