CONSTRUCTION OF A RECOMBINANT PLASMID CONTAINING THE GENE ENCODING CHITOSANASE FROM BACILLUS AMYLOLIQUIFACIENS ABBD AND THE GENE ENCODING RECEPTOR BINDING DOMAIN FROM SARS-COV-2

• Main Author: Choirunnisa, Rinni
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/64662
• Institution: Institut Teknologi Bandung

The SARS-CoV-2 receptor-binding domain (RBD) protein is a target molecule for the treatment and vaccination of the 2019 Coronavirus disease (COVID-19). Recombinant RBD (rRBD) has been produced with Esherichia coli cell systems and with Pichia pastoris cells. The rRBD produced by E. coli was in the form of a protein precipitate, while the rRBD produced by the P. pastoris system was soluble in a limited amount of buffer. To increase the production of rRBD in P. pastoris, the gene encoding rRBD was combined with the gene encoding the chitosanase csn1 which has been shown to have very high expression levels in the P. pastoris cell system. To achieve the aim of this study, the csn1-rbd gene (~1500 base pairs) was constructed using the Polymerase Chain Reaction (PCR) technique and propagated using the pGEM-T Easy vector in E. coli TOP'10F cells. The next process was the subcloning of the csn1-rbd gene into the pPICZ?-A vector on the restriction site EcoRI and XbaI to be expressed in P. pastoris KM71H. The csn1-rbd gene was successfully constructed, propagated, and verified by colony PCR, restriction enzyme analysis, and nucleotide base sequencing analysis using the Sanger method. The nucleotide sequence determination electroferogram showed the size of the csn1-rbd gene was 1347 bp. The recombinant plasmid pPICZ?-A-csn1-rbd was also obtained after confirmation by colony PCR analysis and restriction enzyme analysis. Recombinant pPICZ?-A- csn1-rbd cleavage with EcoRI and XbaI resulted in bands at ~1500 bp and ~3500 bp that correlated with the respective sizes of the csn1-rbd gene and pPICZ?-A plasmid. The results of this study are expected to be used to overcome the problem of recombinant RBD production in P. pastoris.