Recombinant RBD of the SARS-CoV-2 Spike Protein: Production in Escherichia coli Cells, Binding to Antibodies, and Antiviral Activity
MOLECULAR GENETICS MICROBIOLOGY AND VIROLOGY(2023)
摘要
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.
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关键词
SARS-CoV-2,Spike protein,RBD,Escherichia coli expression system,antiviral activity,virus-neutralizing antibodies
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