Construction of Recombinant Plasmids Encoding the sACE2-Fc Gene for the Development of SARS-CoV-2 Neutralization Test
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SARS-CoV-2, sACE2-Fc, Recombinant DNA, DNA rekombinan
Abstract
Abstract
Background: COVID-19 infection is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The presence of neutralizing antibodies in the body of an infected person is necessary to prevent viral infection. The presence of neutralizing antibodies in seroconvalesen or post vaccinated sera can be measured by several techniques. Competitive Elisa using recombinant RBD spike antigens and ACE2 receptors is one of techniques that viable to be developed since this technique can be applied in facility that does not have a BSL 2 facility. In this research was aimed at obtaining a recombinant plasmid that could be used for the production of the soluble ACE2 recombinant (sACE2). To enhanced its activity, the sACE2 was fused to the C-terminal portion of Imunoglobin F (Fc region).
Methods: The sACE2 coding gene was inserted within the NheI and BamHI sites replacing sRBD gene in the pcDNA3-SARS-CoV-2-S-RBD-Fc vector. The presence of sACE2 gene was confirmed using restriction enzyme analysis and sequencing.
Results: The result showed that the recombinant pcDNA3-sACE2(WT)-Fc plasmid was successfully verified using restriction enzymes and sequencing so that it can be used for the production of recombinant soluble ACE2 using mammalian cells.
Conclusions: The construction process of sACE2 into the pcDNA3 SARS-CoV-2-S-RBD-Fc was successfully carried out and verified.
Keywords : SARS-CoV-2, sACE2-Fc, Recombinant DNA
Abstrak
Latar Belakang: Infeksi COVID-19 disebabkan oleh Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Keberadaan antibodi netralisasi dalam tubuh seseorang yang terinfeksi sangat diperlukan untuk mencegah infeksi virus. Antibodi netralisasi dalam serum konvalesen atau serum paska vaksinasi dapat dideteksi dengan beberapa teknik. Elisa kompetitif menggunakan antigen rekombinan RBD spike dan reseptor ACE2 merupakan salah satu teknik yang layak untuk dikembangkan karena teknik ini dapat diterapkan pada fasilitas yang tidak memiliki fasilitas BSL 2. Pada penelitian ini bertujuan untuk mendapatkan plasmid rekombinan yang dapat digunakan untuk produksi rekombinan soluble ACE2 (sACE2). Untuk meningkatkan aktivitasnya, sACE2 digabungkan ke bagian C- terminal dari Imunoglobulin F (Fc region).
Metode: Gen pengkode sACE2 dimasukkan ke dalam situs NheI dan BamHI menggantikan gen S-RBD dalam vektor pcDNA3-SARS-CoV-2-S-RBD-Fc. Keberadaan gen sACE2 dikonfirmasi menggunakan analisis restriksi enzim dan sekuensing.
Hasil: Hasil penelitian menunjukkan bahwa plasmid rekombinan pcDNA3-sACE2(WT)-Fc berhasil diverifikasi menggunakan enzim restriksi dan sekuensing sehingga dapat digunakan untuk produksi rekombinan soluble ACE2 menggunakan sel mamalia.
Kesimpulan: Proses konstruksi sACE2 kedalam plasmid pcDNA3-SARS-CoV-2-S-RBD-Fc telah berhasil dilakukan dan diverifikasi.
Kata kunci : SARS-CoV-2, sACE2-Fc, DNA rekombinan
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Authors
Copyright (c) 2023 Fera Ibrahim, Silvia Tri Widyaningtyas, Devia Puspita Natalicka, Ekawati Betty Pratiwi
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