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Title: The characterisation of Theileria annulata sporozoite surface antigens
Author: Knight, Pamela Anne
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Theileria annulata is a major protozoan parasite of cattle in tropical and subtropical countries, transmitted by the tick Hyalomma anatolicum The infection in the host is initiated by the sporozoite, which rapidly attaches to and invades host cells. A humoral response to sporozoites has been identified in immune cattle (Preston and Brown 1985). Two McAbs which block sporozoite invasion of lymphocytes "in vitro", McAb 1A7 and 4B11, have been identified in previous work (Williamson 1988). These McAbs recognise different sets of polypeptides on Western blots. T. annulata sporozoite antigen 1 (SPAGl), which contains the epitope recognised by McAb 1A7, has been cloned and sequenced [Hall et al 1992). This work describes research to analyse the bovine T and B cell response to the T. annulata SPAGl. The SPAGl polypeptide, and a range of subclones covering different regions of the antigen, were expressed using pGEX vectors in E. coli and purified in large quantities for the purpose of B and T cell epitope mapping. The recombinant SPAGl polypeptides were used to analyse humoral responses of immunised cattle by Western blotting and enzyme linked immuno-sorbant assay. A number of regions of SPAG1 containing B cell epitopes recognised by bovine antisera raised against T. annulata sporozoites were also identified, and the relevance of this data to SPAGl neutralising determinants discussed. T cell responses to SPAGl were analysed in proliferation assays using peripheral blood mononuclear cells and antigen specific T cell lines. A bovine T cell response was found to be induced to SPAG 1 in cattle immunised with the recombinant antigen. In addition, the SPAGl molecule was found to be highly polymorphic according to restriction fragment analysis on Southern blots. Work was also undertaken to prove the surface location of the SPAGl molecule on sporozoites using immuno-electron microscopy techniques. The final part of this work was to identify, express and characterise the T. annulata sporozoite antigen gene containing the McAb 4B11 epitope. McAb 4B11 was used to screen a lambdagtl 1 T. annulata genomic expression library, and two positive recombinants were subcloned into the expression vector pGEX1lambdaT to give pGEX1lambdaT-KP6 and pGEX1lambdaT-KP8. Southern and Northern blot analysis of the KP6 and KPS inserts confirmed they were parasite sequences expressed at high levels in sporozoites as a 3.1 kb mRNA. However, KPS and KPS did not cross-hybridise, indicating that they contained different sequences coding for the 4B11 epitope. The pGEX1lambdaT-KPS fusion protein was expressed in E. coli and purified. The fusion protein was found to affinity bind antibodies from bovine antisera raised against T. annulata sporozoites, and evoked antibodies capable of blocking sporozoite infectivity of leukocytes "in vitro" and recognising sporozoite antigens in immunofluorescence assays or on Western blots when used to immunise a rabbit.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available