RESUMO
BACKGROUND: The Human Cytomegalovirus (HCMV) has infected more than 90% of the world population and its prevalence can be related to the individuals geographical and socialeconomic status. Serological tests based on ELISA are pivotal for HCMV diagnosis. Due to the lack of standardization in the production/purification of antigens from viral preparations, ELISA tests are based on several recombinant proteins or peptides. As an alternative, multiepitope proteins may be employed. OBJECTIVE: In this work, we developed a recombinant multiepitope protein (rMEHCMV) for HCMV diagnosis based on conserved and immunodominant epitopes derived from tegument (pp150, pp65 and pp28), glycoprotein gB (pp38) and DNA polymerase subunit (pp52) of HCMV. METHODS: The rMEHCMV gene was synthesized de novo and overexpressed in Escherichia coli cells. The recombinant protein was purified to homogeneity using a Ni-NTA column. Biophysical analysis of recombinant protein was performed by circular dichroism. A preliminary biological activity test was performed using 12 positive human sera samples by using an in-house IgG ELISA. The following patents database were consulted: Espacenet, Google Patents and the National Institute of Intellectual Property (INPI, Brazil). RESULTS: The recombinant multiepitope protein was successfully expressed in E. coli. The structural data obtained by circular dichroism spectroscopy showed that rMEHCMV is structurally disordered. An in-house IgG ELISA test with rMEHCMV was successfully used to recognized IgG from human serum samples. CONCLUSION: Together, our results show that rMEHCMV should be considered as a potential antigenic target for HCMV diagnosis.
Assuntos
Anticorpos Antivirais , Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/genética , Proteínas Recombinantes , Proteínas Virais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/genética , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
BACKGROUND: Phytases are enzymes capable of degrading phytic acid and are used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus. Recent inventions show interest in production and optimization of recombinant phytases with biochemical and physicochemical characteristics promising for animal feed industry. METHODS: This review article is focused on relevant patents of promising phytases, together with the commonly used expression systems for their production and tools currently employed to generate new phytases. We revised all patents related to recombinant phytases and their application in the animal feed industry. The following patents databases were consulted: European Patent Office (Espacenet), the United States Patent and Trademark Office (USPTO), the United States Latin America Patents (LATIPAT), Patent scope -Search International and National Patent Collections (WIPO) and Google Patents. RESULTS: In this review, information was collected from recent publications, including 38 patents related production systems for different recombinant phytases and their application in the animal feed industry. CONCLUSION: We showed that important recombinant phytases were successfully produced in different expression systems. In addition, this work highlights certain biotechnological tools such as mutagenesis for generation of novel enzymes with biochemical properties of use in the animal feed industry.
