SITE-DIRECTED MUTAGENESIS OF SARS-COV-2 ITB SPIKE DNA FOR DEVELOPMENT OF COVID-19 MRNA VACCINE CANDIDATES

• Main Author: Lilis Fitriani, Annisa
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/78729
• Institution: Institut Teknologi Bandung

COVID-19 mRNA vaccine is one of the types of COVID-19 vaccines still being developed. Compared to other COVID-19 vaccine platforms, mRNA vaccines have safety, efficacy, rapid development, and low-cost manufacturing advantages. COVID-19 mRNA vaccine contains the genetic material mRNA to encode SARSCoV- 2 spike protein. The spike protein (S) is a glycoprotein on the membrane surface of the virus, which plays a role in binding to receptors on host cells and cell membrane fusion. Protein S is metastable to serve its function as a fusion protein that can change the conformations from prefusion to postfusion, which is essential for membrane fusion. These structures are formed through a series of proteolytic activations of the S protein involving the proteases furin and TMPRSS2 that occur during viral biosynthesis and at the host cell surface, respectively. In developing COVID-19 vaccines, spike protein stabilization is essential to create a strong immunity against SARS-CoV-2. Mutations at the furin and TMPRSS2 cleavage sites of the S protein can be carried out to reduce the protein's affinity for proteases, thereby producing a stable S protein structure in the prefusion conformation. R682G and S813Y mutation in the furin and TMPRSS2 cleavage site, respectively, are known to successfully reduce the protein’s affinity for proteases and have a higher potential to generate humoral and cellular immunity against SARS-CoV-2 infection. In this study, site-directed mutagenesis was carried out to change amino acid sequence 682 from arginine (R) to glycine (G) and sequence 813 from serine (S) to tyrosine (Y), which are at the furin and TMPRSS2 cleavage site, respectively, on the DNA encoding the SARS-CoV-2 ITB spike protein. Both mutagenesis were still carried out on separate spike-coding DNA. The site-directed mutagenesis process consists of PCR mutagenesis using mutagenic primers, digestion of nonmutant DNA templates, and transformation into E. coli cells competent for repair of the nicked structure on the plasmid. Mutagenic primer design and testing have been carried out in preparation for PCR mutagenesis. To support further research in developing a COVID-19 mRNA vaccine candidate, a plasmid was constructed to express the SARS-CoV-2 ITB spike mRNA in vitro. Based on the results, the mutagenic primers R682G and S813Y can be used in PCR mutagenesis. The DNA sequencing results of the mutant plasmid showed that the R682G and S813Y mutations, respectively, had been successfully carried out on the DNA encoding the SARS-CoV-2 ITB spike. The plasmid construct for mRNA expression was designed using the pcDNA3.1(+) plasmid backbone and the untranslated region (UTR) derived from the DNA sequence of the ?-globin gene. Mutagenesis at the furin and TMPRSS2 cleavage sites, which are protein proteolytic activation areas, can be utilized as a stabilization strategy for fusion proteins for vaccines against other disease infections.