CLONING OF THE MAJOR OPEN READING FRAMES OF SARS-COV-2, THEIR EXPRESSION IN ESСHERICHIA COLI AND PURIFICATION OF SARS-S-6HIS, SARS-S1-6HIS, SARS-N-6HIS, SARS-M-6HIS AND SARS-E-6HIS PROTEINS

Authors

  • A.V. Zhigailov Almaty Branch of the National Center for Biotechnology, Kazakhstan,
  • Y.O. Ostapchuk M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Kazakhstan, Almaty
  • Y.V. Perfilyeva M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Kazakhstan, Almaty
  • E.R. Maltseva M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Kazakhstan, Almaty
  • Z.A. Berdygulova Almaty Branch of the National Center for Biotechnology, Kazakhstan, Almaty
  • D.A. Naizabayeva M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Kazakhstan, Almaty
  • A.S. Cherusheva M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Kazakhstan, Almaty
  • А.О. Bissenbay Almaty Branch of the National Center for Biotechnology, Kazakhstan, Almaty
  • A.S. Cherusheva Almaty Branch of the National Center for Biotechnology, Kazakhstan, Almaty
  • G.A. Ismagulova M.A. Aitkhozhin’s Institute of Molecular Biology and Biochemistry, Kazakhstan, Almaty
  • А.М. Dmitrovskiy Almaty Branch of the National Center for Biotechnology, Kazakhstan, Almaty
  • Y.A. Skiba Almaty Branch of the National Center for Biotechnology, Kazakhstan, Almaty

DOI:

https://doi.org/10.26577/eb.2023.v94.i1.08

Keywords:

COVID-19, SARS-CoV-2, cloning, recombinant proteins.

Abstract

A novel coronavirus SARS-CoV-2 has been associated with the COVID-19 atypical pneumonia pandemic, which caused a devastating healthcare and socioeconomic crisis worldwide. Effective diagnosis of the disease is important to prevent the spread of infection. Since the detection of antigens and antibodies against SARS-CoV-2 by enzyme-linked immunosorbent assay is of great importance in the diagnosis of COVID-19, we developed recombinant analogs of the main structural proteins of the SARS-CoV-2 virus. The genes of the main open reading frames, including the surface spike (S) glycoprotein, the small membrane (M) protein, the envelope (E) glycoprotein and nucleocapsid (N) protein, were cloned in expression plasmids pET23c in polyhistidine reading frames. We obtained the strains of Esсherichia coli BL-21(DE3) that produce recombinant S-6His, M-6His, E-6His и N-6His proteins. N-6His protein was produced in sufficient quantities, for the purification of which the conditions of dialysis and its concentration were optimized. We obtained pure preparation of N protein (the target protein concentration is 80%). Further studies should be performed to identify its antigenic activity.

References

“COVID-19: Laboratory and diagnosis. Antigen-detection in the diagnosis of SARS-CoV-2 infection using rapid immu- noassays. Interim guidance”. World Health Organization. Retrieved 11 September 2020. https://www.who.int/publications/i/item/ antigen-detection-in-the-diagnosis-of-sars-cov-2infection-using-rapid-immunoassays

“Weekly epidemiological update on COVID-19 – 27 July 2021”. World Health Organization. Retrieved 28 July 2021. https:// www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19 27-july-2021.

“WHO Director-General’s opening remarks at the media briefing on COVID-19 – 11 March 2020”. World Health Organization. 11 March 2020. Retrieved 12 March 2020.

Akin A., Lin T.L., Wu C.C., Bryan T.A., Hooper T., Schrader D. (2001) Nucleocapsid protein gene sequence analysis re- veals close genomic relationship between Turkey coronavirus and avian infectious bronchitis virus. Acta. Virol., vol. 45, pp. 31–38.

Alsharif W., Qurashi A. (2021) Effectiveness of COVID-19 diagnosis and management tools: A review. Radiography (Lond), vol. 27, no 2. pp. 682-687.

Bai Z., Cao Y., Liu W., Li J. (2021) The SARS-CoV-2 Nucleocapsid Protein and Its Role in Viral Structure, Biological Functions, and a Potential Target for Drug or Vaccine Mitigation. Viruses, vol. 13, no 6, pp. 1115.

Baro B., Rodo P., Ouchi D., Bordoy A.E., Saya Amaro E.N., Salsench S.V., et al. (2021) Performance characteristics of five antigen-detecting rapid diagnostic test (Ag-RDT) for SARS-CoV-2 asymptomatic infection: a head-to-head benchmark comparison. J Infect., vol. 82, no 6, pp. 269-275.

Casal J.I., Rodriguez M.J., Sarraseca J., Garcia J., Plana-Duran J., Sanz A. (1998) Identification of a common antigenic site in the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus. Adv. Exp. Med. Biol., vol. 440, pp. 469–477.

Collisson E.W., Pei J., Dzielawa J., Seo S.H. (2000) Cytotoxic T lymphocytes are critical in the control of infectious bron- chitis virus in poultry. Dev. Comput. Immunol., vol. 24, pp. 187–200.

Dutta N.K., Mazumdar K., Gordy J.T. (2020) The Nucleocapsid Protein of SARS-CoV-2: a Target for Vaccine Develop- ment. J. Virol., vol. 94, no 13, e00647-20.

Fehr A.R., Perlman S. Coronaviruses: an overview of their replication and pathogenesis. Coronaviruses. New York: Spring- er, 2015. pp. 1–23.

Hiscox J.A., Caavanagh D., Britton P. (1995) Quantification of individual subgene- mic mRNA species during replication of the coronavirus transmissible gastroenteritis. Virus. Res., vol. 36, pp. 119–130.

Keck J.G., Hogue B.G., Brian D.A., Lai M.M.C. (1988) Temporal regulation of bovine coronavirus RNA synthesis. Virus. Res., vol. 9, pp. 343–356.

La Marca A., Capuzzo M., Paglia T., Roli L., Trenti T., Nelson S.M. (2020) Testing for SARS-CoV-2 (COVID-19): a sys- tematic review and clinical guide to molecular and serological in-vitro diagnostic assays. Reprod. Biomed. Online., vol. 41, no 3, pp. 483-499.

Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, vol. 227, pp. 680-685.

Liao Y., Lescar J., Tam J.P., Liu D.X. (2004) Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability. Biochem. Biophys. Res. Commun., vol. 325, no 1, pp. 374-380.

Oliveira S.C., de Magalhães M.T.Q., Homan E.J. (2020) Immunoinformatic Analysis of SARS-CoV-2 Nucleocapsid Protein and Identification of COVID-19 Vaccine Targets. Front. Immunol., vol. 11, pp. 587615.

Pal M., Berhanu G., Desalegn C., Kandi V. (2020) Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2): An Update. Cureus, vol. 12, no 3, e7423.

Peña M., Ampuero M., Garcés C., Gaggero A., García P., Velasquez M.S., et al. (2021) Performance of SARS-CoV-2 rapid antigen test compared with real-time RT-PCR in asymptomatic individuals. Int J Infect Dis., vol. 107, pp. 201-204.

Sambrook J., Russel D.W. Molecular cloning: A laboratory manual: 3 volumes. – Third edition. New-York: Cold Spring Harbor Laboratory Press, 2001. 2100 pp.

Schoeman D., Fielding B.C. (2019) Coronavirus envelope protein: current knowledge. Virol. J., vol. 16, pp.69.

Tsoi H., Li L., Chen Z.S., Lau K-F., Tsui S.K.W., Chan H.Y.E. (2014) The SARS-coronavirus membrane protein induces apoptosis via interfering with PDK1-PKB/Akt signalling. Biochem. J., vol. 464, pp. 439–447.

ul Qamar M.T., Alqahtani S.M., Alamri M.A., Chen L-L. (2020) Structural basis of SARS-CoV-2 3CLpro and anti-COV- ID-19 drug discovery from medicinal plants. J. Pharm. Anal., vol. 10, no 4, pp. 313-319.

Wage H., Schliephake A., Korner H., Flory E., Wage H. (1993) An immunodomi- nant CD4+ T cells site on the nucleocap- sid protein of murine coronavirus contributes to protection against encephalomyelitis. J. Gen. Virol., vol. 74, pp. 1287–1294.

Yoshimoto F.K. (2020) The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19. Protein J., vol. 39, pp. 198–216.

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Published

2023-03-20

Issue

Vol. 94 No. 1 (2023): Experimental biology

Section

МOLECULAR BIOLOGY AND GENETICS