Purification, Characterization and Vaccine Potential of Trypanosoma brucei brucei Glycosyl Phosphatidyl Inositol Specific Phospholipase C

Abstract

Aims: To investigate possible use of Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) as a target protein for the development of vaccine against Trypanosoma brucei brucei infection was investigated. Study Design: GPI-PLC from T. brucei brucei was purified, characterized and the protein was used as antigen in raising antibody against the parasite Place and Duration: Department of Biochemistry, Ahmadu Bello University Zaria-Nigeria, between September 2011 and October 2012 Methodology: GPI-PLC was isolated from T. brucei brucei and purified by ammonium sulphate precipitation, gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GPI-PLC was further used to raise antisera in rabbits, which was subsequently used to immunize rats for 14 and 21 days pre-infection to investigate the possible use of T. b. brucei GPI-PLC as target protein in vaccine production against T. b. brucei infection. Results: An overall yield of 48.76% and purification fold of 10.86 were recorded after gel filtration. The result from SDS-PAGE showed the enzyme to be a 39.585 kDa protein with optimum temperature, optimum pH and activation energy to be 35ºC, 8.1 and 19.494 kJ/ mol respectively. The Vmax and Km values were 6.67 × 10-3 µmol/hr and 2.67 × 10-3 µM respectively when 212.5 µg of enzyme was used in the reaction mixture. Immunization with anti GPI-PLC for 14 and 21 days pre-infection significantly lowered the Packed Cell Volume (PCV). Result for the time course of parasitemia following infection with 7.9 x 105 Cells/ml showed a decrease in parasitemia level, thus leading to lowering of mortality rates in Groups immunized with GPI-PLC for 14 and 21 days pre-infection by 20% and 40% respectively relative to Group infected but not treated. Conclusion: These results suggest that GPI-PLC as a target protein significantly reduced the progression of the T. b. brucei infection.