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Cloning and Expression of SARS CoV-2 Spike Receptor Binding Domain (RBD) Synthetic Gene in Escherichia coli BL21 (DE3) Expression System

Year: 2024
Type: article
Abstract: To reduce dependence on overseas reagents, a biological test candidate for recombinant proteins must be developed by constructing SARS-CoV-2 receptor binding domain (RBD) target genes. RBD is an outermost protein in SARS-CoV-2 and can recognize cell surface receptors further with the presence of interaction between angiotensin-converting enzyme 2. RBD can cause the occurrence of endocytosis in the cells, which allows for the replication of coronavirus SARS-CoV2, which causes COVID-19 disease. Through research, the aim is to produce recombinant proteins from RBD SARS-CoV-2 using the Escherichia coli BL21 (DE3) expression system. With the availability of recombinant proteins, it is expected to be applied as an antigen used in immunoassay-based diagnostic tests. A total of 969 nucleotides encoding 321 amino acids of SARS-CoV-2 RBD were cloned to the pET-14b vector. The plasmid was then expressed in E. coli BL21 (DE3) and induced with 1.0 mM isopropyl-β-d-1-thiogalactopyranoside. Purification processed and concentrated using PEG-6000 resulted in 0.8-mg/mL protein yield. SARS-CoV-2 RBD recombinant protein analysis using the SDS-PAGE technique showed approximately 35.75 kDa specific band target protein. The results of the study using the ELISA technique on the vaccinated sample showed an average antibody concentration of 0.176 g/mL and on the non-vaccinated samples of 0.062 g/mL. On this basis, it can be stated that the E. coli expression system can be utilized as a biological material in the development of an in-house immunoassay kit for SARS-CoV-2 (COVID-19).
DOI: 10.56899/153.05.06
Authors: Eva Moulia, Uus Saepuloh, Yuliana Yuliana, Huda Shalahudin Darusman
Institution: IPB University
Language: English
Field: Medicine
Open Access status: bronze
OpenAlex ID: W7083690329
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