Title:Rational Design and Evaluation of the Recombinant Multiepitope Protein
for Serodiagnosis of Rubella
Volume: 23
Issue: 8
Author(s): Marilen Souza, Juliana Machado, Jonatas da Silva, Luana Ramos, Lais Nogueira, Patrícia Ribeiro, Daniel Dias, Josiane Santos, José Carlos Santos, Yanna Nóbrega, Amanda Souza, Sonia Freitas, Mariana Campos da Paz, Maria Felipe, Fernando Torres and Alexsandro Galdino*
Affiliation:
- Laboratório de Biotecnologia de Microrganismos, Universidade Federal de São João Del-Rei, Campus Centro-Oeste Dona Lindu, 35501-296, Divinópolis, MG, Brasil
Keywords:
Rubella virus, multiepitope protein, diagnostic test, enzyme immunoassay, fetal abnormalities, glycoprotein.
Abstract:
Background: Rubella is an infection caused by rubella virus (RV) and is generally regarded
as a mild childhood disease. The disease continues to be of public health importance mainly because
when the infection is acquired during early pregnancy, it often results in fetal abnormalities, which are
classified as congenital rubella syndrome (CRS). An accurate diagnosis of rubella is thus of pivotal
importance for proper treatment.
Objectives: The aim of the study was to produce a recombinant multiepitope protein (rMERUB) for
the diagnosis of rubella, based on conserved immunodominant epitopes of glycoprotein E1 and E2.
Methods: A synthetic gene was designed and cloned into vector pET21a with a 6xHis tag at the Cterminal
for affinity purification and overexpressed in Escherichia coli cells. Biophysical analysis of
rMERUB was performed by circular dichroism. Biological activity was assessed using an in-house
ELISA assay.
Results: Expression in Escherichia coli showed a ~22 kDa protein that was purified and used to perform
structural assays and an IgG ELISA. Structural analyses reveal that rMERUB has a β leaf pattern
that promotes the exposure of epitopes, thus allowing antibody recognition. Evaluation of 33 samples
(22=positive; 11=negative) was performed using in-house ELISA and this was compared with a commercial
kit. The sensitivity was 100% (95% CI: 85-100) and specificity 90.91% (95% CI: 62-99).
Excellent agreement (Kappa index = 0.9) was obtained between ELISA assays.
Conclusion: The careful choice of epitopes and the high epitope density, coupled with simple-step
purification, pinpoints rMERUB as a promising alternative for rubella diagnosis, with potential for the
development of a diagnostic kit.
