Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes

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.Keywords: Cryptosporidium, glycoprotein GP60, polyclonal antibody, diagnosis. ResumoNeste trabalho, foi desenvolvido um método de expressão da glicoproteína GP60 de Cryptosporidium hominis em Escherichia coli visando produzir anticorpos IgY anti-GP60 em galinhas para utilização em estudos futuros com os objetivos de diagnóstico, prevenção e tratamento da criptosporidiose. A sequência completa de nucleotídeos do gene gp60 de C. hominis foi códon-otimizada para expressão em E. coli e sintetizada no vetor pET28-a. A subclonagem foi realizada em várias estirpes diferentes de E. coli BL21. Os ensaios de concentração do indutor IPTG, temperatura e tempo foram realizados e analisados por SDS-PAGE. As condições ótimas de expressão foram observadas em temperatura de 37 °C, incubação durante a noite e 1 mM de IPTG. A purificação da proteína foi realizada por cromatografia de afinidade utilizando o sistema de cromatografia AKTA Pure e a coluna Hi-Trap™ HP (GE Healthcare). A proteína recombinante GP60 (rGP60) foi utilizada para imunizar galinhas poedeiras para produzir IgY policlonal anti-rGP60. Verificou-se por Western blot e por imunofluorescência indireta que o anticorpo policlonal apresentou reatividade com a rGP60 e com esporozoítos de Cryptosporidium parvum, respectivamente. A rGP60 e a IgY anti-rGP60 geradas neste estudo podem ser utilizadas como modelos para o desenvolvimento de ensaios para pesquisa e diagnóstico da criptosporidiose.Palavras-chave: Cryptosporidium, glicoproteína GP60, anticorpo policlonal, diagnóstico.

Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review

“…Miura et al produced IgY against the GP60 protein, from the Cryptosporidium hominis protozoan. These IgY bound to GP60 in Western blot and to C. parvum sporozoites in fecal samples by indirect immunofluorescence, suggesting that anti-GP60 IgY could be used in the diagnosis of cryptosporidiosis caused by both C. hominis and C. parvum [116].…”

Immunoglobulin Y for Potential Diagnostic and Therapeutic Applications in Infectious Diseases

“…Using a mouse model, the anti-P23 IgY reduced oocyst shedding by 70% ( 300 ). Specific IgY against the GP60 glycoprotein in Cryptosporidium hominis , another strain implicated in cryptosporidiosis, was also found to specifically bind to the antigen, as well as the parasite ( 302 ). The high specificity and protective effectiveness of these antibodies suggest that IgY warrants evaluation as a novel diagnostic test for cryptosporidiosis and a passive immunization treatment in immunocompromised individuals.…”