The trypanocidal effects of antibodies to T.brucei-recombinant tubulin

David Kateete


T. b. brucei alpha and beta tubulin genes were expressed in E. coli using the pTrP2 expression plasmid and 3-?-Indoleacrylic acid as an inducer. The expressed tubulins were analysed by SDS-PAGE and confirmed by Western blotting, in which they had an identical apparent molecular mass of 50kDa. The expressed tubulins were individually dissolved in 8M Urea and diluted 20-fold, after which they were concentrated and extensively dialyzed against MES buffer. Following dialysis, the average yield for both alpha and beta tubulin was 0.7g per liter of culture. Polyclonal antibodies were produced in each of the 56 rabbits immunized with three doses of the concentrated tubulins at either 150?g or 450?g per immunization per animal. The effect of these antibodies on T. b. brucei growth was studied by culturing T. b. brucei bloodstream form in up to 25% of the rabbit-raised antisera. The antisera from the other rabbits did not inhibit T. b. brucei growth in culture. These results show that antibodies to bacterially expressed trypanosome tubulin may not be cytotoxic to T. b. brucei.

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