Recombinant PFR-like proteins cloned from Trypanosoma cruzi

Chagas disease, or American trypanosomiasis, is a parasitic disease found throughout Central and South America. It is caused by the single-celled parasite,Trypanosoma cruzi, which is transmitted by the reduviid, or kissing bug, a large blood-sucking insect that often lives in rural homes. Trypanosomes have a unique structure, the paraflagellar rod (PFR), which runs along the length of the flagellum. The PFR is composed of a lattice of cytoskeletal filaments and is critical for cell motility. The proteins of the PFR in T. cruzi have been shown to be immunogenic, protecting mice from an otherwise lethal challenge with the parasite. Two previously unidentified PFR-like genes, PFR-5 and PFR-6, were discovered when the T. cruzi genome was sequenced. The aim of this project is to determine if these two putative PFR proteins are associated with the flagellum. Portions of the PFR-5 and PFR-6 genes have been cloned into expression plasmids. These plasmids are expressed in Escherichia coli to produce recombinant proteins which will be harvested, purified, and injected into mice. The mouse immune system will recognize the foreign proteins and respond by producing PFR-5 and PFR-6 antibodies. These antibodies will be harvested and used to determine the subcellular location of the proteins in T. cruzi, using fluorescent markers