CONSTRUCTION AND EXPRESSION OF RBDL452R/T478K-FOLDON FUSION IN PICHIA PASTORIS AS A COVID-19 VACCINE CANDIDATE

• Main Author: Putra, Ahadul
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/64668
• Institution: Institut Teknologi Bandung

Coronavirus Disease (COVID-19) is an infectious disease on the respiratory system caused by SARS-CoV-2. Vaccination is the most appropriate strategy to resolve the COVID-19 pandemic. The receptor-binding domain (RBD), part of the spike (S) protein on the surface of SARS-CoV-2, is directly involved in the interaction with the angiotensin-converting enzyme (ACE2) receptor on the host cells. Therefore, RBD becomes an ideal target for vaccine candidates. The Delta variant which had two mutations on RBD namely L452R and T478K had the ability to interact more strongly with ACE2. Therefore, it was necessary to make a Delta variant of the RBD antigen. Immunogenicity enhancement can be carried out by producing an RBD trimer with foldon protein derived from fibritin protein in bacteriophage. The aim of this research was to construct the RBD-Foldon coding gene containing mutations in the L452R and T478K coding codons and then determined the optimum conditions for RBDL452R/T478K-Foldon expression. The construction of pPICZ?A- RBDL452R/T478K-F was carried out by directed mutagenesis method using PCR. The mutated sequences were confirmed by DNA sequencing. Integration of RBDL452R/T478K-Foldon was performed by homologous recombination. The RBDL452R/T478K-Foldon protein was characterized using SDS-PAGE and Western Blot. The pPICZ?-RBDL452R/T478K-F plasmid was successfully constructed. The ability of P. pastoris X-33 to grow on YPD-zeocin with various concentrations showed that P. pastoris X-33 genome had RBDL452R/T478K-Foldon. ~1.3kb and ~2.2kb of DNA fragment bands generated by PCR confirmed that the RBDL452R/T478K-Foldon gene was indeed integrated into the P. pastoris X-33 genome and the AOX1 gene was not disrupted. Expression of RBDL452R/T478K-Foldon gene by 3 transformants with 2% methanol as the inducer for 48 hours showed the presence of a band measuring ~73 kDa on SDS-PAGE which was predicted as the molecular weight of RBDL452R/T478K-Foldon trimer. The result of western blot analysis confirmed that bands were RBDL452R/T478K-Foldon trimer which interacted with the antibody of RBD SARS-CoV-2. The optimum of induction duration, methanol concentration, aeration, and initial cell density (OD600) for production of the RBDL452R/T478K-Foldon protein was for 3 days, 3% of methanol, 10% of aeration, and OD600 60 respectively.