Abstract

The aim of the study is to synthesize a recombinant protein in Escherichia coli cells that carries the receptor-binding domain (RBD) of Spike protein of SARS CoV-2, with antiviral activity comparable to the activity of RBD obtained in a eukaryotic cells (eRBD). 6His-RBD_short (24.4 kDa) and 6His-RBD_long (33.7 kDa) proteins were expressed in Escherichia coli strain BL21 (DE3). Chromatographic purification of the proteins was carried out on WorkBeads 40 Ni-NTA and WorkBeads 40S sorbents followed by multistage refolding. Enzyme immunoassay was performed using GamP2C5 and GamXRH19 humanized single-domain monoclonal antibodies specific for SARS-CoV-2 Spike protein RBD. Antiviral activity against the SARS-CoV-2 virus was studied using Vero E6 cells. 6His-RBD_short recombinant protein was synthesized in Escherichia coli cells, including the Spike protein RBM (receptor-binding motif) of SARS-CoV-2 virus (330–527 а.а.). Two-stage chromatographic purification of 6His-RBD_short recombinant protein was performed, followed by refolding. Enzyme immunoassay demonstrated effective interaction of 6His-RBD_short recombinant protein with virus neutralizing antibodies, comparable to eRBD. The study of antiviral activity showed inhibition of SARS-CoV-2 virus reproduction after treatment of Vero E6 cells with 6His-RBD_short (45.1%) and eRBD (42.8%) proteins. The 6His-RBD_long recombinant protein obtained in the same work, which included a longer fragment of RBD, did not interact with virus neutralizing antibodies and did not inhibit the replication of the SARS-CoV-2 virus. After conducting additional studies, the developed 6His-RBD_short recombinant protein can be considered a promising drug for therapeutic use as an ACE2 receptor blocker.

中文翻译：

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SARS-CoV-2 刺突蛋白的重组 RBD：大肠杆菌细胞中的产生、与抗体的结合以及抗病毒活性

摘要

该研究的目的是在大肠杆菌细胞中合成一种重组蛋白，该蛋白携带SARS CoV-2刺突蛋白的受体结合域（RBD），其抗病毒活性与在真核细胞中获得的RBD活性相当（eRBD） ）。6His-RBD_短(24.4 kDa) 和 6His-RBD_{长(33.7 kDa) 蛋白在}大肠杆菌中表达菌株 BL21 (DE3)。在 WorkBeads 40 Ni-NTA 和 WorkBeads 40S 吸附剂上对蛋白质进行色谱纯化，然后进行多级重折叠。使用针对 SARS-CoV-2 Spike 蛋白 RBD 的 GamP2C5 和 GamXRH19 人源化单域单克隆抗体进行酶免疫测定。使用 Vero E6 细胞研究了针对 SARS-CoV-2 病毒的抗病毒活性。在大肠杆菌细胞中合成了6His-RBD_短重组蛋白，包括 SARS-CoV-2 病毒 (330–527 а.а.) 的刺突蛋白 RBM（受体结合基序）。_{对 6His-RBD短}重组蛋白进行两阶段色谱纯化，然后进行重折叠。_{酶联免疫分析证明 6His-RBD短链}有效相互作用具有病毒中和抗体的重组蛋白，与 eRBD 相当。抗病毒活性研究表明，用 6His-RBD_短蛋白 (45.1%) 和 eRBD (42.8%) 蛋白处理 Vero E6 细胞后，可抑制 SARS-CoV-2 病毒繁殖。同一工作中获得的6His-RBD长重组蛋白，包含较长的RBD片段，不与病毒中和抗体相互作用，并且不抑制SARS-CoV-2病毒的复制_。经过进一步的研究，所开发的 6His-RBD_短重组蛋白可被认为是一种有前景的 ACE2 受体阻滞剂治疗药物。