Purification of H1N1 virus and screening of its specific ssDNA aptamers using GO-SELEX

다국어 초록 (Multilingual Abstract)

The emergence of new influenza A viruses generated by frequent genetic mutations causes the pandemic worldwide ever year. Among them, H1N1 has spread over many countries and caused considerable number of infections, deaths and substantial economic damages since its first outbreak in Mexico in 2009. Thus, early detection of the H1N1 virus is very important to prevent these losses. In this study, for accurate and sensitive detection of H1N1 virus, the virus solution was purified by dialysis method, and the purified virus solution was used for the aptamer selection. The purity of virus solution was 89.3%. After 10 rounds of Graphene Oxide-based Systematic Evolution of Ligands by Exponential Enrichment (GO-SELEX) including 2rounds of counter selection to improve specificity of aptamers to the target, 13 ssDNA aptamer candidates that can bind to H1N1 virus were selected. For the selected aptamer candidates, pre-screening test and characterization were carried out by Graphene Oxide based-Fluorescence Resonance Energy Transfer (GO-FRET) assay. Among 13 candidates, HS1 and HS5 showed high affinity and specificity to the target virus than the other candidates. The dissociation constants (Kd value) of HS1 and HS5 were calculated as 4.2 x 106 PFU/ml and 9.2 x 106 PFU/ml, respectively.

목차 (Table of Contents)

• Table of contents
• LIST OF FIGURES ......................................................................Ⅲ
• ABBREVIATIONS .......................................................................Ⅴ
• ABSTRACT .................................................................................Ⅵ
• Ⅰ. INTRODUCTION .....................................................................1
• Ⅱ. MATERIALS AND METHODS ...............................................5
• 2.1. Materials and chemicals .................................................................5
• 2.2. Purification of H1N1 influenza A virus in allantoic fluid using dialysis method and confirmation of morphological integrity of purified H1N1 virus via TEM image for target preparation .................6
• 2.2.1. Dialysis method .........................................................................6
• 2.2.2. Bradford assay ..........................................................................7
• 2.2.3. Negative staining of purified H1N1 virus for TEM image .......8
• 2.3. Selection of H1N1 influenza A virus-specific ssDNA aptamers by using GO-SELEX .............................................................................9
• 2.3.1. Normal SELEX ...........................................................................9
• 2.3.2. Counter SELEX .......................................................................10
• 2.3.3. Cloning and Sequencing ..........................................................11
• 2.4. Characterization of H1N1 influenza A virus-specific ssDNA aptamers via GO-FRET assay ...........................................................12
• 2.4.1. Pre-screening test .................................................................12
• 2.4.2. Dose-dependency test & Specificity test .............................13
• Ⅲ. RESULTS ..............................................................................14
• 3.1. Purification of H1N1 influenza A virus using dialysis method ...14
• 3.2. Screening of aptamer candidates by using GO-SELEX .............19
• 3.3. Characterization of aptamer candidates via GO-FRET assay ...23
• Ⅳ. DISCUSSION .........................................................................34
• Ⅴ. CONCLUSION .......................................................................36
• Ⅵ. REFERENCES .......................................................................37
• ABSTRACT IN KOREAN ...........................................................43
• ACKNOWLEDGEMENT .............................................................44