Designing structure and E. coli strain-producer bearing SARS-CoV-2 N, S, M, E protein-related sequence antigen

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Abstract

T-cell immune response is extremely important in protecting human body from diverse viral infections. It is known that it can ensure viral clearance and complete recovery in patients with humoral immunodeficiency. COVID-19 patients were found to have T-cell response primarily directed against SARS-CoV-2 structural S, M, N, E proteins, with nucleocapsid protein being most conserved. To assess patients’ immunity against coronavirus infection and evaluate an effectiveness of vaccine candidates, it is necessary to develop an optimal diagnostic antigen to evaluate arising T-cell response against SARS-CoV-2 antigenic determinants. A diagnostic test to determine host specific susceptibility to SARS-CoV-2 infection should target conserved regions of global SARS-CoV-2 variants. The study was aimed to develop a structure of an antigen bearing conserved and immunogenic sequences derived from SARS-CoV-2 structural proteins and to obtain an Escherichia coli producer strain containing a recombinant protein to be subsequently used for assessing antiviral T-cell immunity. Developing of the antigen was performed in silico: TepiTool and NetMHCIIpan were used to predict and identify high affinity epitopes spanning SARS-CoV-2 E, M, N, S proteins and MHC II binding. 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, as well as 3D representation built by using the I-TASSER. The sequence was synthesized and cloned into the pET24a(+) vector. The resulting plasmid pCorD_PS was transformed into E. coli DH5α followed by Rosetta (DE3). The strain-producer of the recombinant E. coli protein CorD_PS was assessed for the presence and stability of IPTG-induced antigen protein expression and elimination of recombinant coronavirus antigen-bearing plasmid. Based on the study data, an antigen was developed consisting of conserved regions from SARS-CoV-2 S, M, N, E proteins. A 53 kDa recombinant protein was predicted to be stable in aqueous solutions with isoelectric point of 9.56 potentially allowing to simplify protein purification from E. coli cells. Plasmid DNA pCorD_PS (6695 bp) encoding final recombinant coronavirus antigen cloned into pET24a(+) vector was obtained. A stable, productive E. coli CorD_PS strain was obtained. The obtained strain-producer resulting in recombinant E. coli CorD_PS antigen is stable allowing to move on to design antigen purification technique and further develop SARS-CoV-2-specific diagnostic test system.

About the authors

Vladimir V. Kopat

LLC “ATG Service Gene”

Email: kopat@service-gene.ru
ORCID iD: 0000-0002-6573-6743

Development Director

Russian Federation, 199178, St. Petersburg, int. ter. municipal district Vasilyevsky, Maly pr. V.O., 57, build. 4, letter Zh, room 5-N, office 1.2.5

Anastasia A. Riabchenkova

LLC “ATG Service Gene”

Author for correspondence.
Email: riabchenkova@service-gene.ru
ORCID iD: 0000-0002-9973-0753

Researcher

Russian Federation, 199178, St. Petersburg, int. ter. municipal district Vasilyevsky, Maly pr. V.O., 57, build. 4, letter Zh, room 5-N, office 1.2.5

Evgenii L. Chirak

LLC “ATG Service Gene”

Email: chirak.evgenii@service-gene.ru
ORCID iD: 0000-0001-9167-5000

Researcher

Russian Federation, 199178, St. Petersburg, int. ter. municipal district Vasilyevsky, Maly pr. V.O., 57, build. 4, letter Zh, room 5-N, office 1.2.5

Elizaveta R. Chirak

LLC “ATG Service Gene”

Email: chirak.elizaveta@service-gene.ru
ORCID iD: 0000-0002-1610-8935

Researcher

Russian Federation, 199178, St. Petersburg, int. ter. municipal district Vasilyevsky, Maly pr. V.O., 57, build. 4, letter Zh, room 5-N, office 1.2.5

Anna I. Saenko

LLC “ATG Service Gene”

Email: anna.saenko@gmail.com
ORCID iD: 0009-0003-1059-1991

Chief Process Engineer

Russian Federation, 199178, St. Petersburg, int. ter. municipal district Vasilyevsky, Maly pr. V.O., 57, build. 4, letter Zh, room 5-N, office 1.2.5

Nikolai N. Kolmakov

Institute of Experimental Medicine

Email: kolmakov@service-gene.ru
ORCID iD: 0000-0002-4672-6208

Researcher, Department of Molecular Genetics

Russian Federation, Saint Petersburg

Andrey S. Simbirtsev

Saint Petersburg Pasteur Institute

Email: simbas@mail.ru
ORCID iD: 0000-0002-8228-4240

RAS Corresponding Member, DSc (Medicine), Professor, Head of the Laboratory of Medical Biotechnology

Russian Federation, 197101, Saint Petersburg , st. Mira, 14

Ilya V. Dukhovlinov

LLC “ATG Service Gene”

Email: atg@service-gene.ru
ORCID iD: 0000-0002-5268-9802

PhD (Biology), Director of Science

Russian Federation, 199178, St. Petersburg, int. ter. municipal district Vasilyevsky, Maly pr. V.O., 57, build. 4, letter Zh, room 5-N, office 1.2.5

Areg A. Totolian

Saint Petersburg Pasteur Institute

Email: totolian@spbraaci.ru
ORCID iD: 0000-0003-4571-8799

RAS Full Member, DSc (Medicine), Professor, Director

Russian Federation, 197101, Saint Petersburg , st. Mira, 14

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Supplementary files

Supplementary Files
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1. JATS XML
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2. Figure 1. Linear structure of the chimeric protein

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3. Figure 2. Modeled structure of the recombinant coronavirus antigen (3D protein model) rendered with I-TASSER [31]

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4. Figure_3. Schematic representation of the pCorD_PS recombinant vector construction

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5. Figure 4. Electropherogram of the restricted vector pCorD_PS in 1% agarose gel

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6. Figure 5. Antigen expression in cultures of 3 clones induced with the addition of 1 mM IPTG in 12% polyacrylamide gel under denaturing conditions

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7. Figure 6. Electropherogram of recombinant coronavirus antigen expression after 1–9 passages

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8. Figure 7. Electropherogram of plasmid DNA after successive passages

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9. Figure 8. Electropherogram of plasmid DNA restriction after successive passages

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Copyright (c) 2023 Kopat V.V., Riabchenkova A.A., Chirak E.L., Chirak E.R., Saenko A.I., Kolmakov N.N., Simbirtsev A.S., Dukhovlinov I.V., Totolian A.A.

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