Submit Manuscript  

Article Details


A Custom-Designed Recombinant Multiepitope Protein for Human Cytomegalovirus Diagnosis

[ Vol. 13 , Issue. 4 ]

Author(s):

Patrícia A.F. Ribeiro, Marilen Q. Souza, Daniel S. Dias, Alice C. M. Álvares, Laís M. Nogueira, Juliana M. Machado, José C. dos Santos, Renato R. Godoi , Yanna K.M. Nobrega, Mariana Campos-da-Paz, Sonia M. de Freitas, Maria S.S. Felipe, Fernando A.G. Torres and Alexsandro S. Galdino*   Pages 316 - 328 ( 13 )

Abstract:


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.

Keywords:

Human cytomegalovirus, recombinant, multiepitope protein, diagnostic test, enzyme immunoassay, diagnosis.

Affiliation:

Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinopolis, MG, 35501-296, Departamento de Biologia Celular, Universidade de Brasilia, Brasília, DF, 70910-900, Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinopolis, MG, 35501-296, Laboratorio de Biofisica, Universidade de Brasilia, Brasilia, DF, 70910-900, Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinopolis, MG, 35501-296, Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinopolis, MG, 35501-296, Departamento de Biologia Celular, Universidade de Brasilia, Brasília, DF, 70910-900, Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinopolis, MG, 35501-296, Laboratorio de Doencas Imunogeneticas e Cronico-degenerativas, Universidade de Brasilia, Brasilia, DF, 70910-900, Laboratorio de Nanobiotecnologia, Universidade Federal de Sao Joao Del-Rei, Divinopolis, MG, 35501-296, Laboratorio de Biofisica, Universidade de Brasilia, Brasilia, DF, 70910-900, Departamento de Biologia Celular, Universidade de Brasilia, Brasília, DF, 70910-900, Departamento de Biologia Celular, Universidade de Brasilia, Brasília, DF, 70910-900, Laboratorio de Biotecnologia de Microrganismos, Universidade Federal de Sao Joao Del-Rei, Divinopolis, MG, 35501-296

Graphical Abstract:



Read Full-Text article