Ekspresi Gen Sintetik Receptor Binding Domain Spike SARS-COV-2 Pada Sistem Ekspresi Escherichia coli

Abstract

Receptor Binding Domain (RBD) merupakan bagian dari spike SARS-Cov-2 yang secara spesifik dapat berikatan dengan reseptor ACE-2 (Angiotensin converting enzyme-2). Penelitian ini bertujuan untuk menghasilkan protein rekombinan dari RBD SARS-Cov-2 menggunakan sistem ekspresi Escherichia coli BL21 (DE3). Ketersediaan protein rekombinan ini diharapkan dapat diaplikasikan sebagai antigen untuk mengembangkan uji diagnostik berbasis immunoassay. Perancangan gen sintetik RBD menggunakan cetakan urutan nukleotida pada Genbank MZ306692.1 yang dioptimasi kodonnya dan dilengkapi dengan sisi pemotongan enzim restriksi XhoI dan BamHI untuk selanjutnya disisipkan ke vector pET-14b. Gen sintetik RBD kemudian diekspresikan menggunakan E. coli BL21 dan diinduksi menggunakan 0,1mM IPTG (Isopropyl B-D-1-thiogalactopyranoside). Pemurnian protein rekombinan dilakukan dengan teknik kolom kromatografi afinitas Ni2+ - NTA dan dianalisis hasil ekspresinya menggunakan teknik SDS-PAGE. Sistem pemurnian Ni2+-NTA berhasil menghilangkan beberapa protein non-spesifik dan menghasilkan 0,8 mg/mL protein rekombinan RBD SARS Cov-2 murni dan menunjukkan pita protein spesifik berukuran 35,75 kDa. Aplikasi protein rekombinan RBD SARS Cov-2 murni pada teknik ELISA dengan direaksikan terhadap sampel yang divaksinasi menunjukkan nilai rata-rata konsentrasi antibodi sebesar 0,176 g/mL dan pada sampel yang tidak divaksinasi sebesar 0,062 g/mL. Hasil penelitian ini menunjukkan bahwa protein rekombinan RBD SARS CoV-2 yang dikembangkan dalam sistem ekspresi E. coli dapat digunakan sebagai bahan biologis untuk kit deteksi immunoassay in-house terhadap SARS COV-2 (COVID-19).

Receptor binding domain (RBD) is a part of the S protein in SARS CoV-2 that specifically binds to the ACE-2 (Angiotensin-converting enzyme-2) receptor. This study aims to express a recombinant protein of RBD spike SARS-CoV-2 using Escherichia coli BL21 (DE3). The availability of this recombinant protein is expected to apply an antigen to develop immunoassay-based diagnostic tests. The RBD SARS CoV-2 nucleotide sequences were obtained from the GenBank database of MZ306692.1 which was optimized for codon and equipped with the cleavage site of the XhoI and BamHI restriction enzymes for further insertion into the pET-14b vector, expressed in E. coli BL21 (DE3), and then induced with 1.0 mM IPTG. The recombinant protein purification was performed using the Ni2+-NTA affinity chromatography column technique resulting in 0.80 mg/mL of purified RBD SARS CoV-2 recombinant protein. The expression and refining analyses were conducted using SDS-PAGE and have discovered the successfully recombinant protein with a specific band measuring approx ~36 kDa. This purified recombinant protein has been applied to the ELISA technique using vaccinated and non-vaccinated samples and indicated significant results, such as a vaccinated concentration mean of 0.176 µg/mL and a non-vaccinated concentration mean of 0.062 µg/mL. The constructed RBD SARS CoV-2 gene was successfully expressed and purified. Therefore, the E. coli expression system could be used as an alternative to scale up the production of SARS CoV-2 RBD recombinant protein. This RBD SARS CoV-2 recombinant protein was developed to contribute in-house biological materials to produce an immunoassay detection kit for the SARS COV-2 (COVID-19).