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Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application

Abstract Introduction: Chikungunya virus (CHIKV) causes febrile illnesses in human and these cases have rapidly expanded across the globe in recent years. The current antibody-based tests for CHIKV such as ELISA have a variety of limitations associated with the molecules such as batch-to-batch vari...

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Main Authors: Anna, Andrew, Magdline Sia, Henry Sum, Citartan, Marimuthu, Thean-Hock, Tang
Format: Proceeding
Language:English
Published: 2021
Subjects:
QR355 Virology
Online Access:http://ir.unimas.my/id/eprint/36849/1/ICMBB%202021%20Abstract%20submission%20form_FINAL.pdf
http://ir.unimas.my/id/eprint/36849/
https://themsmbboffice.wixsite.com/4thicmbb2021
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spelling my.unimas.ir.368492021-12-01T08:50:23Z http://ir.unimas.my/id/eprint/36849/ Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application Anna, Andrew Magdline Sia, Henry Sum Citartan, Marimuthu Thean-Hock, Tang QR355 Virology Abstract Introduction: Chikungunya virus (CHIKV) causes febrile illnesses in human and these cases have rapidly expanded across the globe in recent years. The current antibody-based tests for CHIKV such as ELISA have a variety of limitations associated with the molecules such as batch-to-batch variation, high cost and less stable. Aptamers are single-stranded DNA or RNA that have high affinity and specificity against a wide variety of target molecules. Compared to antibodies, aptamers are cheaper, produced in vitro, no batch-to-batch variations and thus serve as a good molecular recognition element for the development of diagnostic tests for CHIKV. Methods: Cloning, expression and purification of the recombinant CHIKV E2 was carried out and its identity was verified with western blot analysis. The purified protein was subjected to 9 SELEX cycles, the resulting nucleic acid pools were cloned and sent for sequencing. The secondary structure of the aptamer was predicted using Mfold web server and the performance of the aptamer was determined by enzyme-linked aptamer assay (ELAA). Result: The 24kDa recombinant E2 proteins were successfully cloned and purified. The protein was reactive against anti-CHIKV positive sera and anti-CHIKV polyclonal antibody with no cross reactivity with anti-dengue positive pool sera. Sequencing result revealed there were 6 potential candidates of DNA aptamers. DNA aptamer candidate with the highest frequency (61.9%) showed two loops in their predicted secondary structures. ELAA analysis revealed a binding affinity (Kd) of 177.5 nM and limit of detection was 3.3 nM. Conclusion: DNA aptamers were successfully generated and it has great potential as a feasible tool in CHIKV detection. Keywords: Chikungunya virus, E2 protein, DNA aptamer, SELEX 2021-06-01 Proceeding PeerReviewed text en http://ir.unimas.my/id/eprint/36849/1/ICMBB%202021%20Abstract%20submission%20form_FINAL.pdf Anna, Andrew and Magdline Sia, Henry Sum and Citartan, Marimuthu and Thean-Hock, Tang (2021) Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application. In: 4th International Conference on Molecular Biology and Biotechnology 2021 (ICMBB2021), 1st -3rd June 2021, Virtual Conference. https://themsmbboffice.wixsite.com/4thicmbb2021
institution Universiti Malaysia Sarawak
building Centre for Academic Information Services (CAIS)
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Sarawak
content_source UNIMAS Institutional Repository
url_provider http://ir.unimas.my/
language English
topic QR355 Virology
spellingShingle QR355 Virology
Anna, Andrew
Magdline Sia, Henry Sum
Citartan, Marimuthu
Thean-Hock, Tang
Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application
description Abstract Introduction: Chikungunya virus (CHIKV) causes febrile illnesses in human and these cases have rapidly expanded across the globe in recent years. The current antibody-based tests for CHIKV such as ELISA have a variety of limitations associated with the molecules such as batch-to-batch variation, high cost and less stable. Aptamers are single-stranded DNA or RNA that have high affinity and specificity against a wide variety of target molecules. Compared to antibodies, aptamers are cheaper, produced in vitro, no batch-to-batch variations and thus serve as a good molecular recognition element for the development of diagnostic tests for CHIKV. Methods: Cloning, expression and purification of the recombinant CHIKV E2 was carried out and its identity was verified with western blot analysis. The purified protein was subjected to 9 SELEX cycles, the resulting nucleic acid pools were cloned and sent for sequencing. The secondary structure of the aptamer was predicted using Mfold web server and the performance of the aptamer was determined by enzyme-linked aptamer assay (ELAA). Result: The 24kDa recombinant E2 proteins were successfully cloned and purified. The protein was reactive against anti-CHIKV positive sera and anti-CHIKV polyclonal antibody with no cross reactivity with anti-dengue positive pool sera. Sequencing result revealed there were 6 potential candidates of DNA aptamers. DNA aptamer candidate with the highest frequency (61.9%) showed two loops in their predicted secondary structures. ELAA analysis revealed a binding affinity (Kd) of 177.5 nM and limit of detection was 3.3 nM. Conclusion: DNA aptamers were successfully generated and it has great potential as a feasible tool in CHIKV detection. Keywords: Chikungunya virus, E2 protein, DNA aptamer, SELEX
format Proceeding
author Anna, Andrew
Magdline Sia, Henry Sum
Citartan, Marimuthu
Thean-Hock, Tang
author_facet Anna, Andrew
Magdline Sia, Henry Sum
Citartan, Marimuthu
Thean-Hock, Tang
author_sort Anna, Andrew
title Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application
title_short Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application
title_full Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application
title_fullStr Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application
title_full_unstemmed Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application
title_sort generation of dna aptamers against envelope 2 (e2) protein of chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (selex) for diagnostic application
publishDate 2021
url http://ir.unimas.my/id/eprint/36849/1/ICMBB%202021%20Abstract%20submission%20form_FINAL.pdf
http://ir.unimas.my/id/eprint/36849/
https://themsmbboffice.wixsite.com/4thicmbb2021
_version_ 1718930118455328768
score 13.211869

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