ABSTRACT
The Nsp1 protein, the main virulence factor of the virus of SARS-CoV-2, mediates viral immune escape in host cells and expands the viral infection range. Systematic bioinformatics analysis and prokaryotic expression of Nsp1 protein were performed to aid the understanding of the molecular mechanism of SARS-CoV-2 infection in host cells. Pfam, TMHMM, ProtScale, ExPASy, SignalP 4.0 and other tools were used to systematically analyze the post-translational modifications, physicochemical properties, transmembrane helix, interaction network, homology and evolutionary characteristics of Nsp1 protein. The recombinant expression vector pET-22b-Nsp1 was constructed through molecular cloning technology and expressed in a prokaryotic system. Nsp1 is composed of 180 amino acids, with a molecular weight of 19.78 kDa, an isoelectric point of 5.36 and an instability index of 28.83. It has a half-life of 30 h in mammalian reticulocytes and more than 10 h in E. coli. It has 12 potential phosphorylation sites, three potential O-glycosylation sites and no signal peptide. It is a hydrophilic protein without a transmembrane helix. Secondary structure analysis indicated that the highest proportion of structural components comprised random coils (45.00%), followed by a-helices (25.56%) and extended chains (20.56%);the lowest proportion comprised ss-turns (8.89%). Multiple sequence alignment and evolutionary analysis revealed that Bat SARS-like coronavirus WIV1 had the highest sequence identity (85.00%) with the Nsp1 protein of SARS-CoV-2. After prokaryotic expression, Nsp1 protein was found to be mainly expressed in the precipitate after centrifugation of bacterial lysate. The target protein was further identified as Nsp1 by mass spectrometry. This study provides an important reference for the expression, purification and functional analysis of SARS-CoV-2 Nsp1 protein, and further reveals the biological functions of Nsp1, thus providing a reference for research and development of related inhibitors and antiviral drugs.