ABSTRACT
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.
Resumo Neste 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.
Subject(s)
Animals , Female , Immunoglobulins/immunology , Chickens/immunology , Cryptosporidiosis/diagnosis , Cryptosporidium/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/chemistry , Cryptosporidiosis/immunology , Escherichia coli/metabolismABSTRACT
Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of Ca2+, Mg2+, K+, and HCO3 - in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.
Subject(s)
Animals , Humans , Mice , ATP-Binding Cassette Transporters/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Calcium/metabolism , Cloning, Molecular , Cryptosporidiosis/parasitology , Cryptosporidium/chemistry , Iron/metabolism , Molecular Sequence Data , Protein Structure, Tertiary , Protozoan Proteins/chemistry , Sequence AlignmentABSTRACT
En este trabajo se evaluó la aplicabilidad de la técnicas de tinción de Kinyoun e Inmunofluorescencia, para el monitoreo periódico de la calidad parasitológica del agua residual del Sistema de Lagunas de Estabilización "Planta de Tratamiento de Aguas Servidas Sur" de la ciudad de Maracaibo. Se detectó Cryptosporidium sp. en el 100 por ciento de las muestras analizadas por las dos técnicas. Los promedios de Cryptosporidium sp. detectados por la técnica de Kinyoun fueron: en la entrada de la planta 4,9x105 ooquistes/100L, en los módulos primarios 1,2x105 ooquistes/100L, en la laguna facultativas 5,9x104 ooquistes/100L y en la salida 3,0x104 ooquistes/100L, mientras que para C. parvum por inmunofluorescencia fueron: 5,9x104 ooquistes/100L, 7,7x104 ooquistes/100L, 3,0x105 ooquistes/100L y 3,0x104 ooquistes/100L, respectivamente. Se observó una correspondencia del 100 por ciento de positividad en las muestras analizadas por ambas técnicas. Dada las ventajas en facilidad de aplicación, rapidez y costos que ofrece la técnica de Kinyoun, se sugiere su aplicación como técnica de rutina para la evaluación de Cryptosporidium sp. en muestras de agua residual. Se observó la presencia de par sitos en el efluente final, por lo tanto éste no es apto desde el punto de vista parasitológico, para ser empleado con fines de irrigación, sin representar un riesgo para la salud pública.
This work evaluates the applicability of the Kinyoun and immunofluorescence techniques for periodic control of the parasitological quality of wastewater from the stabilization lagoon system Wastewater Treatment Plant South in the city of Maracaibo. Cryptosporidium sp. was detected in the 100 percent of the samples analyzed by both techniques. The concentration of Cryptosporidum sp. detected by the Kinyoun technique was: 4.9x105 oocysts/100L at the plant entrance, 1.2x105 oocysts/100L in the primary modules, 5.9x104 oocysts/100L in the facultative lagoon and 3.0x104 oocysts/100L in the final effluent, while those of C. parvum detected by inmunofluorescence were: 5.9x104 oocysts/100L, 7.7x104 oocysts/100L, 3.0x105 oocysts/100L and 3.0x104 oocysts/100L, respectively. A 100 percent correspondence of the results from samples analyzed by both techniques was observed. Given the advantages in ease of application, rapidity and costs offered by the Kinyoun technique, its routine application for evaluating Cryptosporidium sp. in wastewater samples is suggested. The presence of parasites in the final effluent was observed; therefore, from the parasitological viewpoint, it is not apt for irrigation purposes, without representing a risk to human health.