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

• Grant number: 13/05622-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2013
• End date: September 30, 2016
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Preventive Veterinary Medicine
• Principal Investigator: Marcelo Vasconcelos Meireles
• Grantee: Alex Akira Nakamura
• Host Institution: Faculdade de Medicina Veterinária (FMVA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil
• Associated scholarship(s): 15/14315-9 - Super expression of COWP6 protein from Cryptosporidium parvum aiming the production of monoclonal IgG in mice for diagnostic purpose, BE.EP.PD

Abstract

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)