CONSTRUCTION AND EXPRESSION OF PET16B-RBD-FOLDON PLASMID

• Main Author: Sekar Ayu Citraningsukma, Nimas
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/62838
• Institution: Institut Teknologi Bandung

COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in 486,761,597 cases and 6,142,735 deaths worldwide by March 26, 2022, according to World Health Organization (WHO). SARS-CoV-2 enters host cells through the interaction of Spike protein (Protein S) of the virus with Angiotensin Converting Enzyme 2 (ACE-2) human receptor. Receptor Binding Domain (RBD) of Protein S plays an important role in ACE-2 binding. RBD is able to induce immune response to produce neutralizing antibodies. Therefore, RBD is a promising SARS-CoV-2 vaccine candidate to prevent SARS-CoV-2 infection. It has been shown that the RBD trimer has higher immunogenicity compared to RBD monomer. The aims of this study were to undertake an in silico study to generate a model structure of RBD fused with foldon domain of T4 fibritin of bacteriophage, as well as to construct a recombinant plasmid containing a gene encoding the RBD-Foldon and to express the recombinant RBD-Foldon in Escherichia coli BL21(DE3). Trimer RBD-Foldon trimer modeling performed using Alphafold2 server resulted in a good quality model as indicated by Predicted Local Distance Difference Test (plDDT) and Predicted Aligned Error (pAE) parameters. The pET16b_RBD-Foldon recombinant plasmid has been generated and was verified by colony PCR, restriction enzyme analysis, and nucleotide sequence analysis. SDS-PAGE analysis showed that RBD-Foldon was expressed as a monomer with a molecular mass of ~26 kDa in E. coli BL21(DE3) under experimental condition. Further studies are required to find optimum condition which allows formation of RBD- Foldon trimer.