The identification of aptamers against serum biomarkers of human tuberculosis
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Date
2018
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Publisher
University of the Western Cape
Abstract
Tuberculosis (TB) is a global health problem and rated as the second leading cause of death after HIV/AIDS. Transmission of TB from one person to the next is very rapid in crowded communities. Therefore, it is crucial to identify people who are infected as quickly as possible not only to provide treatment but also to prevent the spread of the disease. Current TB diagnostic tests such as the culture and sputum smear tests are time-consuming, while rapid tests make use of antibodies that are costly and have low sensitivity and stability. Great improvement has been observed when aptamers are used in place of antibodies in rapid diagnostic tests such as lateral flow devices (LFDs). Therefore, the current study aims to synthesize and identify aptamers against serum biomarkers for development of rapid TB diagnostic tests such as a lateral flow assay. Several TB serum biomarkers have been identified and can be used for the diagnosis of TB.
TB biomarkers expressed in serum samples were identified through in silico approach. The biomarkers were expressed in bacterial systems using recombinant DNA technology. The recombinant proteins were purified by affinity chromatography and further used as targets for the selection of aptamers using Systemic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers for the selected biomarkers were synthesized based on magnetic-bead based SELEX and characterized by electrophoretic mobility shift assay (EMSA), Surface Plasmon resonance (SPR) and MicroScale Thermophoresis (MST).
Six putative TB serum biomarker proteins were selected from literature, namely, Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Interferon-stimulated Gene 15 (ISG15), Calcium Binding Protein (S100A9), Retinol Binding Protein 4 (RBP4), Granzyme A (GrA), and Transgelin-2 (TAGLN2).
The biomarkers were recombinantly expressed and purified after which they were used as targets in SELEX for aptamers synthesis. Aptamers were analysed by in silico method and the ones with highly conserved motifs were selected. The selected aptamers were synthesized and later characterized. The aptamers that show high affinity and specificity for the biomarkers will be used for the fabrication of a rapid lateral flow device for TB screening. Such a test would allow for a short diagnostic turnaround time, and hence expedite treatment.
Description
>Magister Scientiae - MSc
Keywords
Tuberculosis, Recombinant DNA technology, Electrophoretic Mobility Shift Assay (EMSA), Aptamers, In silico approach