RESUMO
GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 µg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays.
Assuntos
Anticorpos Monoclonais/biossíntese , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Glutationa Transferase/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Schistosoma japonicum/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Avidina/química , Biotina/química , Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/biossíntese , Glutationa Transferase/genética , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Hibridomas/imunologia , Hibridomas/patologia , Limite de Detecção , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Schistosoma japonicum/enzimologia , Baço/citologia , Baço/imunologiaRESUMO
Several papers on Schistosoma mansoni and Schistosomajaponicum proteomes have been published worldwide in the past few years, representing an emerging field of study. Knowledge of the schistosome proteome may greatly enhance our understanding of schistosome physiological processes at the molecular level, and may provide new models for development of vaccines or drugs. Despite the importance of this approach, schistosome proteomic research in Brazil is still incipient. Here we review the development of schistosome proteomic research around the world and provide an appreciation on the future opportunities in Brazil.