COVID-19 mRNA Aşı Geliştirilmesinde Kullanılacak Olan Yüksek Konsantrasyondaki Plazmid DNA’sının Optimizasyonu: Alkalen Lizis Metodu ve Ticari Kit Sonuçlarının Kıyaslanması

Abstract

While forming the stable IVT mRNA molecule, high concentration and purity plasmid DNA must be obtained to ligase the ORF antigen sequence initially copied from the plasmid DNA with the UTR regions. In this study, in the stage of creating the mRNA molecule, which is the first step of the COVID-19 mRNA vaccine, comparison and optimization of the pDNA containing the ORF target antigen sequence were performed as a result of isolation with alkaline lysis method and commercial kit. Plasmid DNA bacteria containing the target antigen ORF sequence were grown under appropriate conditions. Plasmid DNA was isolated by commercial kit and alkaline lysis method from bacterial cultures stopped at different OD600 nm values (0.02-0.05, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5). After the obtained pDNAs were visualized on an agarose gel, their purity and concentration were measured by spectroscopic measurement. After the stab culture is resuscitated in SOC medium, bands are formed in a single form after isolation with the kit, and in multiple forms (linear, supercoiled, circular) after pDNA isolation by alkaline lysis method. The ideal OD600 nm for both methods was 0.3-0.4. As a result of isolation with the kit, higher purity on the contrary low concentration pDNA was obtained. The ideal OD600 nm value is a critical parameter that affects the concentration and purity of pDNA. The alkaline lysis method is a cheap and powerful technique that can be used as an alternative for mRNA vaccine development compared to kit isolation.