Recombinant Antigen Expression and Purification

Recombinant TS was expressed in bacteria and purified as described elsewhere [3 (link)]. CLCP [47 (link)], KMP-11 [41 (link), 42 (link)] and Tol-T [40 (link)] encoding genes were cloned by PCR performed on genomic T. cruzi DNA and cloned into pTrcHis (Invitrogen) to be expressed in Escherichia coli BL21. Proteins were purified by immobilized metal affinity chromatography through Ni²⁺-charged Hi-Trap chelating columns (GE-Healthcare) to immunize BALB/cJ mice. The central and variable region (nucleotides 109 to 390) of one TcMUCII gene (GenBank U32448.1) was cloned by PCR into pGEX-1λT vector (GE Healthcare). GST-TcMUCII protein was expressed and purified by affinity chromatography and used for immunization. The protocol of animal immunization followed in this study was approved by the Committee on the Ethics of Animal Experiments of the Universidad Nacional de San Martín (UNSAM), according with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.