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Rational Design and Evaluation of the Recombinant Multiepitope Protein for Serodiagnosis of Rubella
Marilen Souza1, Juliana Machado2, Jonatas da Silva2
1Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Universidade de Brasília, Brasília, 70910-900, Brasília, DF, Brasil.
Insights
A new recombinant multiepitope protein (rMERUB) shows promise for accurate rubella diagnosis. This protein, derived from rubella virus glycoproteins, achieved 100% sensitivity in initial tests, offering a potential new diagnostic tool.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- Rubella virus (RV) infection is typically mild but poses significant risks during pregnancy, causing congenital rubella syndrome (CRS).
- Accurate rubella diagnosis is crucial for effective management and prevention of CRS.
Purpose of the Study:
- To develop a recombinant multiepitope protein (rMERUB) for improved rubella diagnosis.
- The rMERUB is based on conserved, immunodominant epitopes from rubella virus glycoproteins E1 and E2.
Main Methods:
- A synthetic gene encoding rMERUB was designed, cloned into a pET21a vector with a 6xHis tag, and expressed in Escherichia coli.
- Purified rMERUB underwent biophysical analysis (circular dichroism) and biological activity assessment via an in-house ELISA.
Main Results:
- The expressed protein (~22 kDa) exhibited a beta-sheet structure, facilitating epitope exposure for antibody recognition.
- In an in-house IgG ELISA, rMERUB demonstrated 100% sensitivity and 90.91% specificity in evaluating 33 samples, with excellent agreement (Kappa=0.9) compared to a commercial kit.
Conclusions:
- The rMERUB, characterized by strategic epitope selection and high epitope density, offers a promising alternative for rubella diagnosis.
- This recombinant protein has the potential for the development of a novel diagnostic kit for rubella.
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.
