Abstract
The development of recombinant vaccines against SARS-CoV-2 is required to eliminate the COVID-19 pandemic. We reported the expression of a recombinant protein Flg-RBD comprising receptor binding domain of SARS-CoV-2 spike glycoprotein (RBD) fused to flagellin of Salmonella typhimurium (Flg), known as mucosal adjuvant, in Nicotiana benthamiana plants. The fusion protein, targeted to the cytosol, was transiently expressed using the self-replicating vector pEff based on potato virus X genome. The recombinant protein Flg-RBD was expressed at the level of about 110–140 μg per gram of fresh leaf tissue and was found to be insoluble. The fusion protein was purified using metal affinity chromatography under denaturing conditions. To increase the yield of Flg-RBD, the flow-through fraction obtained after loading of the protein sample on the Ni-NTA resin was re-loaded on the sorbent. The yield of Flg-RBD after purification reached about 100 μg per gram of fresh leaf tissue and the purified protein remained soluble after dialysis. The control flagellin was expressed in a soluble form and its yield after purification was about 300 μg per gram of fresh leaf biomass. Plant-produced Flg-RBD protein could be further used for the development of intranasal recombinant mucosal vaccines against COVID-19.
Highlights
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The pEff vector contains the 50 -untranslated fragment of the potato virus X (PVX) genome, the gene encoding an RNA-dependent RNA polymerase enabling the replication of the vector in a plant cell, promoter of the first viral subgenomic RNA, AMV translation enhancer (50 nontranslated region of RNA 4 of the alfalfa mosaic virus), the gfp gene flanked by unique
Due to the presence of RNA-dependent RNA polymerase gene, the vector can self-replicate in a plant cell, which ensures a high level of expression of the target protein
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The emergence of new pathogenic strains of viruses is a constant threat to human health throughout the world. The Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) is a novel coronavirus that caused an ongoing pandemic in the human population. An outbreak of coronavirus disease 2019 (COVID-19) was reported in December 2019 in the city of Wuhan, Hubei province of China. The COVID-19 pandemic has rapidly spread all over the world and has put global public health at high risk [1].
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