Volume 9, Issue 3 (2018)
JMBS 2018, 9(3): 317-323 |
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Rashedi H, Arjmand S, Rashedi H, Ranaei Siadat S, Pouryaqubi M. Detection of DNA Aptamer with High Affinity against Hepatitis B Surface Antigen by Systematic Evolution of Ligands by Exponential Enrichment. JMBS 2018; 9 (3) :317-323
URL: http://biot.modares.ac.ir/article-22-24336-en.html
URL: http://biot.modares.ac.ir/article-22-24336-en.html
1- Biotechnology Department, Chemical Engineering Faculty, University of Tehran, Tehran, Iran
2- Protein Research Center, Shahid Beheshti University, Tehran, Iran
3- Biotechnology Department, Energy & New Technologies Faculty, Shahid Beheshti University, Tehran, Iran, Protein Research Center, Shahid Beheshti University, G. C., Tehran, Iran, Postal code:1983969411 , o_ranaei@sbu.ac.ir
4- Nanobiotechnology Department, Biological Science Faculty, Tarbiat Modares University, Tehran, Iran
2- Protein Research Center, Shahid Beheshti University, Tehran, Iran
3- Biotechnology Department, Energy & New Technologies Faculty, Shahid Beheshti University, Tehran, Iran, Protein Research Center, Shahid Beheshti University, G. C., Tehran, Iran, Postal code:1983969411 , o_ranaei@sbu.ac.ir
4- Nanobiotechnology Department, Biological Science Faculty, Tarbiat Modares University, Tehran, Iran
Abstract: (9062 Views)
Aims: Hepatitis B is a viral infection, which can cause serious liver problems. Hepatitis B surface antigen (HBsAg), which is produced as recombinant, is used to produce the Hepatitis B vaccine. The aim of this study was to detect DNA aptamer with high affinity against HBsAg by Systematic Evolution of Ligands by Exponential Enrichment (SELEX).
Materials and Methods: In the present experimental study, SELEX method was used to isolate and sequence a DNA aptamer with high affinity against HBsAg. The affinity of this monoclonal nucleotide sequence was calculated by fluorimetric method. The difference of initial absorption and residual value as a measure for the number of associated sequences were calculated with Prism 5 software by nonlinear regression method, Binding-saturation and one site-total model were performed, and the amount of electron affinity (Kd) was determined.
Findings: After performing the SELEX procedure and evaluating the amplified sequence with agarose gel, the result was positive control sample containing a bond in the range of 72nucleotides, indicating successful amplification of the selected sequence, using selective primers. During cloning steps from existing colonies of PCR reaction with aptamer specific primers, the presence of aptamer was confirmed in Escherichia coli bacteria. The reported aptamer had a stable secondary structure with a free energy of ΔG of less than -6.9kJ and Tm higher than 45°C.
Conclusion: The selected DNA aptamer has a high affinity to the target protein (HbsAg) and can be considered as an alternative for mAbs in chromatography column.
Materials and Methods: In the present experimental study, SELEX method was used to isolate and sequence a DNA aptamer with high affinity against HBsAg. The affinity of this monoclonal nucleotide sequence was calculated by fluorimetric method. The difference of initial absorption and residual value as a measure for the number of associated sequences were calculated with Prism 5 software by nonlinear regression method, Binding-saturation and one site-total model were performed, and the amount of electron affinity (Kd) was determined.
Findings: After performing the SELEX procedure and evaluating the amplified sequence with agarose gel, the result was positive control sample containing a bond in the range of 72nucleotides, indicating successful amplification of the selected sequence, using selective primers. During cloning steps from existing colonies of PCR reaction with aptamer specific primers, the presence of aptamer was confirmed in Escherichia coli bacteria. The reported aptamer had a stable secondary structure with a free energy of ΔG of less than -6.9kJ and Tm higher than 45°C.
Conclusion: The selected DNA aptamer has a high affinity to the target protein (HbsAg) and can be considered as an alternative for mAbs in chromatography column.
Subject:
Agricultural Biotechnology
Received: 2016/04/18 | Accepted: 2016/07/17 | Published: 2018/09/22
Received: 2016/04/18 | Accepted: 2016/07/17 | Published: 2018/09/22
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