In vitro selection of RNA aptamers that specifically bind to 50kDa outer membrane protein of salmonella enterica serovar typhi
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Date
2016-02
Authors
Ramli, Siti Mariam Mawarni
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Typhoid fever is a food borne illness caused by the bacteria, Salmonella enterica serovar Typhi (S. Typhi). Typhoid fever remains a public health problem in many of the underdeveloped and developing countries including Malaysia. It has been estimated around 22 million cases and 216,000 related deaths occurred worldwide annually. The 50 kDa outer membrane protein (OMP) of S. Typhi has been considered as a possible candidate that plays role during the infection of typhoid. Therefore 50kDa OMP of S. Typhi was used as target molecule in detecting of S. Typhi. Currently, monoclonal and polyclonal antibodies were widely used in diagnostic kit development for antigen detection from the organisms that caused the disease. However, this diagnostic kits are not suitable to be used in the fields because of the antibody are not stable at high temperature and need to maintain the temperature chain. Thus, as a new approach, RNA aptamers were selected for S. Typhi antigen detection in the diagnosis of typhoid fever. Compare to antibody, a few properties of aptamer such as more stable at high temperature make this aptamer more suitable to be used in diagnostic technology, where it can substitute the antibody in the future. Aptamer is a synthetic, single stranded nucleic acid that folds up into a unique two or three-dimensional structure, allowing them to bind specifically to the target molecules. The aptamer was generated using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. SELEX is a process for screening specific ligands from large libraries of oligonucleotides by an iterative process of selection and amplification. SELEX demonstrated six potential aptamer candidates that dominated the final pool of oligonucleotides. The alignment analysis showed the consensus sequences GUU, GUUU, and GUUUU occurred in most of the aptamer candidates which could be the potential binding site of the aptamers towards target
molecule. The binding activity between the aptamers and the target protein was evaluated by electrophoretic mobility shift assays (EMSA). From the six aptamer candidates isolated, only aptamer ST01, ST03 and ST04 showed binding affinity towards 50 kDa OMP of the S. Typhi. Since 50 kDa OMP of S. Typhi is in a complex formation, comprising 3 subunits, subunit 1, marker flagellin protein, subunit 2, glycerol kinase and subunit 3 TolC protein, the three selected aptamers were further tested against the subunits individually. The results showed that aptamers ST01 and ST03 bound to both subunits 2 and 3, while ST04 could not bind to any subunits. As a conclusion, aptamers ST01 and ST03 can bind to the 50 kDa OMP complex and its subunits individually and ST04 would only bind to the 50 kDa OMP complex. These three aptamers, ST01, ST03 and ST04 have the potential to be used as high affinity ligands for the capture and subsequently detection of S. Typhi in the diagnosis of typhoid fever.
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Keywords
Typhoid fever remains , a public health problem.