CONSTRUCTION, EXPRESSION, AND CHARACTERIZATION OF BANN RBDN331Q IN PICHIA PASTORIS
Coronavirus disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. According to World Health Organization (WHO), more than 775 million COVID-19 cases have been reported as of 28 April 2024, with over 6,8 million of them from Indon...
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| Format: | Final Project |
| Language: | Indonesia |
| Subjects: | |
| Online Access: | https://digilib.itb.ac.id/gdl/view/81409 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Coronavirus disease 2019 (COVID-19) is a disease caused by severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2) infection. According to World Health
Organization (WHO), more than 775 million COVID-19 cases have been reported as of 28
April 2024, with over 6,8 million of them from Indonesia. SARS-CoV-2 infection starts
with the binding of the receptor-binding domain (RBD) of the viral spike (S) protein with
angiotensin-converting enzyme 2 (ACE2) receptor on the cell membrane. The infection can
be prevented by vaccination with RBD protein that can induce the production of
neutralization antibody. Some researches have shown that RBD multimerization can
increase vaccine immunogenicity. One way to achieve multimer formation is by fusing
RBD with ?-annulus (Bann) polypeptide from tomato bushy stunt virus (TBSV) capsid.
RBD has two glycosylation sites, N331 and N343. N343 glycosylation plays an important
role in maintaining the active conformation of RBD, while N331 glycosylation could pose
steric hindrance to Bann-RBD multimerization. The purposes of this research are to do site
directed mutagenesis (SDM) to produce Bann-RBDN331Q variant, construction of Pichia
pastoris X-33 carrying Bann-RBDN331Q-encoding gene, expression of Bann-RBDN331Q
encoding gene, and characterization of Bann-RBDN331Q protein. Mutation of N331
encoding codon has been done and verified by nucleotide sequencing. P. pastoris X-33
carrying Bann-RBDN331Q-encoding gene has been constructed and verified by PCR.
Expression of Bann-RBDN331Q-encoding gene in P. pastoris was induced by the addition of
2% methanol. SDS-PAGE analysis showed the presence of protein with a molecular mass
of around 34 kDa. Characterization with endoglycosidase Hf (Endo Hf) showed that the
protein is glycosylated indicated by decrease of molecular mass. Characterization with
enzyme-linked immunosorbent assay (ELISA) showed that the produced Bann-RBDN331Q
could interact with S1 IgG human monoclonal antibody. The results of this research showed
that Bann-RBDN331Q could be a candidate of COVID-19 vaccine |
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