Abstract
In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.
Highlights
Human cryptosporidiosis is caused predominantly by Cryptosporidium hominis and Cryptosporidium parvum (CACCIÒ & POZIO, 2006; XIAO & RYAN, 2004)
Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to recombinant protein GP60 (rGP60) and to Cryptosporidium parvum sporozoites, respectively
Several genetic markers are used for specific identification of Cryptosporidium, the most commonly used locus for subtyping in molecular epidemiology studies is the gp60 gene, which encodes the glycoprotein GP60
Summary
Human cryptosporidiosis is caused predominantly by Cryptosporidium hominis and Cryptosporidium parvum (CACCIÒ & POZIO, 2006; XIAO & RYAN, 2004). A method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.
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