Pachebat, Justin Alexander; (2002) Merozoite surface protein-7. A novel protein on the surface of Plasmodium falciparum merozoites. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

During the erythrocytic cycle of human malaria caused by Plasmodium falciparum, merozoites released from mature schizonts attach to and invade red blood cells. Understanding this invasion process may identify suitable chemotherapy or vaccine targets with which to combat malaria. On the merozoite surface is a complex of polypeptides derived by proteolytic cleavage from merozoite surface protein-1 (MSP-1), and two unrelated 36kDa and 22kDa polypeptides derived from MSP-6 and MSP-7 respectively. On erythrocyte invasion the majority of the MSP-1 complex is shed into the blood stream. This shed complex contains MSP-636, MSP-722, and a 19kDa polypeptide (MSP-719) proposed to be the product of N-terminal cleavage of MSP-722. MSP-1 is a major vaccine candidate, hence any proteins complexed to it, such as MSP-722, are potential vaccine targets and warrant further research. To study MSP-722 and its relationship with MSP-1, it was necessary to clone the msp-7 gene. A combination of genome database searches and Vectorette PCR steps were used to obtain the gene coding for MSP-7, and confirmed that MSP-719 is related to MSP-722. RT-PCR was used to examine msp-7 for the presence of introns, and the precursor's 351 amino acid sequence analysed using structure and function prediction programmes. The msp-7 gene was cloned and sequenced from several lines of P. falciparum and found to be highly conserved. To obtain antibodies with which to study MSP-7 biosynthesis, GST-fusion proteins were produced using bacterial expression systems, and used to immunise mice, producing anti-sera and a specific monoclonal antibody. The pattern of MSP-7 transcription throughout the erythrocytic cycle was analysed by Northern blotting. The translation of 48 kDa MSP-7 precursor and subsequent processing events were determined by western blotting, immunofluorescence and immunoprecipitation techniques. These suggested that the MSP-7 precursor associated with the MSP-1 precursor in schizonts and subsequently underwent three proteolytic processing steps. The time and kinetics of MSP-1 and MSP-7 precursor complex formation, and processing events, were analysed by immunoprecipitation, pulse chase and sensitivity to Brefeldin A. Preliminary experiments were performed to determine the relationship between MSP-1 and MSP-7 processing in merozoites.

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