The result of OX40L-Fc fusion protein plasmid assem-

Abstract

Background: Recent studies investigating the efÏcacy of immunotherapy against malignancies and the consequential importance of immune modulation have elucidated the promising potential of immunotherapeutic interventions in combatting cancer. Activation of the OX-40 pathway has been identified as a pivotal mechanism in augmenting T-cell effector functions. To this end, two primary classes of OX-40 agonists have been harnessed: anti-OX40 monoclonal antibodies and OX40L-Fc fusion proteins. Notably, OX40L-Fc fusion proteins offer distinct advantages over anti-OX40 monoclonal antibodies, particularly in terms of production simplicity, thus holding promise for potential implementation within the biomedical landscape of Mongolia. The impetus for this study is derived from these foundational insights. Aim: Assembling the OX40L-Fc fusion protein plasmid using recombinant DNA technology Materials and method: The design of the plasmid blueprint was conducted utilizing SnapGene software, adhering to the specifications outlined in US patent US20160024176A1 for MEDI6383 OX40L-Fc fusion protein. Synthesis of DNA sequences ensued in accordance with the Polymerase Chain Assembly (PCA) technique as per established protocols. Subsequently, ligation of vector sequences and synthesized inserts was performed employing HindIII and XhoI restriction enzymes. Following this, purification of the resultant plasmids and digested vectors was executed, with the integrity of the constructs being validated via agarose gel electrophoresis. Subsequent to assembly, Gibson assembly methodology facilitated seamless fusion, with confirmation of resulting plasmids accomplished through PCR amplification and restriction enzyme digestion. Results: The application of the Gibson assembly technique yielded the synthesis of a plasmid clone in accordance with the prescribed design parameters. Upon scrutiny, the cloned entity exhibited the integrated sequences of IgG4Fc, Traf, and OX40L, as intended. Subsequent validation via restriction enzyme analysis afÏrmed the successful integration of the targeted fragments into the construct. Conclusion: Upon validation through both Polymerase Chain Reaction (PCR) analysis and restriction enzyme digestion, the plasmid was confirmed to have been effectively assembled.