Produksi Antibodi Poliklonal Menggunakan Protein Rekombinan RBD-spike Untuk Deteksi SARS-CoV-2
Abstract
Abstrak
SARS-CoV-2 merupakan virus yang menyebabkan Coronavirus Disease 2019 (COVID-19) di seluruh dunia dan sampai saat ini kasus terbaru masih terus dilaporkan. Diagnostic test merupakan hal yang krusial untuk dikembangkan. Prinsip diagnostic test COVID-19 berbasis antigen, yaitu mendeteksi virus SARS-CoV-2 melalui respon antibodi dari penderita. Penelitian ini bertujuan untuk memproduksi antibodi poliklonal menggunakan protein rekombinan RBD-Spike untuk mendeteksi virus SARS-CoV-2 berbasis antibodi. Penelitian dimulai dengan penentuan domain RBD-Spike menggunakan pensejajaran asam amino, dan konstruksi DNA untuk RBD-Spike pada vektor ekspresi pET28a menggunakan sintetik nukleotida. Produksi protein rekombinan RBD-Spike diekspresikan pada sel bakteri Escherichia coli. Purifikasi dilakukan untuk memperoleh protein RBD-Spike dan selanjutnya digunakan sebagai antigen untuk induksi antibodi poliklonal pada kelinci. Hasil penelitian menunjukkan bahwa ekspresi protein rekombinan RBD-Spike SARS-CoV-2 memerlukan induksi IPTG 0,1 mM dan terekspresi dalam bentuk inclusion bodies dengan ukuran 39 kDa. Purifikasi protein RBD-Spike dilakukan menggunaan resin afinitas NiNTA, elektroelusi, dan dialisis. Total protein RBD-Spike yang diperoleh sebanyak 4 mL dengan konsentrasi 10 mg/mL. Analisa Ouchterlony menunjukkan bahwa antibodi poliklonal terdeteksi pada minggu kedua setelah injeksi booster dan analisa spesifitas antibodi terhadap antigen menunjukkan bahwa antibodi poliklonal dapat mendeteksi protein RBD-Spike pada konsentrasi 0,1 µg. Selanjutnya diharapkan antibodi poliklonal dapat digunakan untuk deteksi keberadaan virus SARS-CoV-2 dan dapat dikembangkan untuk kit deteksi berbasis antibodi.
Abstract
SARS-CoV-2 is the virus that causes Coronavirus Disease 2019 (COVID-19) worldwide and the latest cases are still being reported until now. The diagnostic test is a crucial to be developed. The principle of the antigen-based COVID-19 diagnostic test is to detect the SARS-CoV-2 virus through antibody response from the patients. This study was conducted to produce polyclonal antibodies using recombinant protein RBD-Spike. The research was carried out by determining the RBD-Spike domain using amino acid alignment and constructing the DNA of RBD-Spike to the expression vector of pET28a using nucleotide synthesis. Production of RBD-Spike recombinant protein was expressed in Escherichia coli. Purification was carried out to obtain RBD-Spike protein and used to induce polyclonal antibody ina rabbit. The results showed that the expression of RBD-Spike recombinant protein required induction of IPTG 0.1 mM and was expressed in inclusion bodies with molecular size of 39 kDa. The purification of RBD-Spike protein was carried out using resin affinity, electroelution, and dialysis. The total protein of RBD-Spike obtained was 4 mL with a concentration of 10 mg/mL. Ouchterlony analysis revealed that polyclonal antibody was detected in the second week after booster injection and analysis of antibody specificity showed that polyclonal antibodies detected RBD-Spike protein at the concentration of 0.1µg of RBD-Spike protein. Moreover, it is expected that our polyclonal antibody detect the SARS-CoV-2 virus and can be developed for antibody-based detection kits.
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DOI: https://doi.org/10.15408/kauniyah.v16i2.25664 Abstract - 0 PDF - 0
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