Development of a structure and strain-producer E. coli of an antigen containing sequences of N, S, M, E proteins of the SARS-COV-2 coronavirus

Abstract

Introduction. T-cell immune response is important in protecting the human body from many viral infections. It is known that it can provide viral clearance and complete recovery in patients with humoral immunodeficiency. In patients with COVID-19, the T-cell response is mainly directed to the structural S, M, N, E proteins of SARS-CoV-2, of which the nucleocapsid protein is the most conservative. To assess the immunity of patients against coronavirus infection and evaluate the effectiveness of vaccine candidates, it is necessary to develop an optimal diagnostic antigen used to assess the formation of a T-cell response against antigenic determinants of SARS-CoV-2. A diagnostic test to determine the specific susceptibility of an organism to SARS-CoV-2 infection should target conserved regions of SARS-CoV-2 global variants. The aim. To develop a structure of an antigen containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus, and obtaining a strain - Escherichia coli - a producer of a recombinant protein for subsequent use of the protein as an antigen for assessing T-cell antiviral immunity. Materials and methods. Developing of the antigen was performedin silico: TepiTool and NetMHCIIpan were used to predict and identify high affinity epitopes spanning SARS-CoV-2 E, M, N, S proteins and binding MHC II. Several variants of recombinant antigen proteins were constructed, from which one was selected based on its physicochemical properties: isoelectric point, hydrophobicity index and aliphatic index, and a 3D representation built using the I-TASSER. The sequence was synthesized and cloned into the pET24a(+) vector. The resulting plasmid pCorD_PS was transformed intoE. coliDH5, then into Rosetta (DE3). The strain-producer of the recombinantE. coliprotein CorD_PS was checked for the presence and stability of the expression of the antigen protein by IPTG induction, and the elimination of the plasmid encoding the synthesis of the recombinant coronavirus antigen was also evaluated. Results. As the result of the research an antigen has been developed that includes conserved regions of the S, M, N, E proteins of the SARS-CoV-2 coronavirus, to which a T-cell immune response can form. For a 53 kDa protein, stability in aqueous solutions and an isoelectric point of 9.56 are predicted, which will potentially simplify the process of protein purification fromE. colicells. Plasmid DNA pCorD_PS (6695 bp) encoding synthesized recombinant coronavirus antigen cloned into pET24a(+) vector was obtained. Conclusion. A stable, productive producing strain ofE. coliCorD_PS was obtained.The obtained strain-producer of the recombinantE. coliCorD_PS antigen is stable, which makes it possible to move on to the creation of an antigen purification technique and the subsequent development of a diagnostic test system.