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Expression and purification of Cryptosporidium hominis GP60 and Cryptosporidium parvum COWP6 recombinant proteins aiming the production of polyclonal IgY in chickens for diagnostic purpose


Cryptosporidium spp. are protozoa which infect a wide variety of vertebrates. Cryptosporidium parvum has many animal hosts, including humans, and is a major etiological agent of diarrhea in ruminants. Cryptosporidium hominis is a species adapted to the human host. Among the various genetic markers that are used for specific identification of Cryptosporidium, the locus most commonly used for molecular epidemiology studies is the gene that encodes the glycoprotein GP60; this locus contains several regions with high mutation rates, is associated with the adhesion of the parasite to the host cell, and is a potential candidate for the development of new drugs, vaccines and new diagnostics methods and treatment for cryptosporidiosis. The Cryptosporidium oocysts wall proteins (COWP) are a group of proteins located on the surface of the oocyst wall and are involved in its structure and resistance of oocysts in environmental conditions. GP60 and COWP proteins are potential targets for cloning and heterologous expression in E. coli, for the production of recombinant proteins useful for the development of diagnostic methods like enzyme immunoassay (ELISA) and direct immunofluorescence assay (DIA), and also for isolation and purification of oocysts through immunological techniques. The aims of this study are the cloning, expression and purification of the recombinant proteins GP60 of C. hominis and COWP6 of C. parvum, followed by immunization of laying hens in order to obtain IgY anti-GP60 and IgY anti-COWP6. After obtaining polyclonal IgY, it will be accomplished the development of capture ELISA for detection of soluble antigens (GP60) of C. hominis and the DIA for detection (COWP6 protein) of C. parvum oocysts. (AU)

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