Rational design and evaluation of the recombinant multiepitope protein for serodiagnosis of rubella

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 for rubella is thus of pivotal importance for proper treatment. Objective: 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 C-terminal 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 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. Conclusions: 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.