Heterologous expression of invariant surface glycoproteins, ISG75 of Trypanosoma brucei brucei and T.b. gambiense, for antibody production and diagnosis of African Trypanosomiasis.
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Date
2013
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Abstract
Accurate diagnosis of the presence of an infectious organism is very important for therapeutic interventions and consequently the recovery of the individual. There is a need for identifying new diagnostic antigens for the serological diagnosis of trypanosomiasis, a disease of humans and animals in Africa caused by protozoa belonging to the genus Trypanosoma. Invariant surface glycoproteins (ISGs) are present in most strains of the parasite and have the potential to replace the variable surface glycoproteins as diagnostic antigens. In order to avoid the challenges of in vivo culturing of bloodstream form (BSF) trypanosomes in laboratory animals, ISG65 and ISG75, the two most common ISGs were heterologously expressed in Escherichia coli and Pichia pastoris expression systems.
The extracellular domains of ISG65 and ISG75 of both T. b. brucei and T. b. gambiense were amplified by PCR from genomic DNA using appropriate primers to give inserts of 1121 bp and 1342 bp sizes. These were sub-cloned into the pGEX-4T1 and pET28a expression vectors. Chemically competent E. coli BL21 (DE3) were transformed using the resultant plasmids and the transformed E. coli cells were used for heterologous protein expression.
The expressed proteins were purified by three phase partitioning (TPP), nickel or glutathione affinity and molecular exclusion chromatography and analysed by reducing SDS-PAGE. The glycosylation status of ISG65 and ISG75 expressed in the M5 strain of P. pastoris, which has an engineered N-glycosylation pathway that produces glycosylated proteins similar to what is obtained in trypanosomes, was determined. The enzymatic action of Endoglycosidase H resulted in a shift in the electrophoretic migration of ISG65 but not ISG75 on SDS-PAGE, confirming N-glycosylation.
Anti-ISG65 and anti-ISG75 antibodies were produced in chickens and affinity purified using the respective recombinant proteins immobilised on affinity matrices. The antibodies recognised native ISG65 and ISG75 respectively in western blots of lysates of T. b. brucei parasites cultured in vitro. Similar recognition of the native ISGs by the anti-recombinant ISG antibodies was also obtained using immunofluorescence microscopy of fixed T. b. brucei parasites. The results obtained demonstrate the potential of application of the recombinant ISG65 and ISG75 and their respective antibodies in the diagnosis of African trypanosomiasis.
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Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.
Keywords
African trypanosomiasis--Diagnosis., Trypanosoma brucei., Immunoglobulins., Theses--Biochemistry.