Email this article Optimization of purification conditions and study of antigenic properties of recombinant nucleocapsid protein of different SARS-CoV-2 strains
- Authors: Rak A.Y.1, Donina S.A.1, Isakova-Sivak I.N.1, Rudenko L.G.1
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Affiliations:
- Institute of Experimental Medicine
- Issue: Vol 22, No 2 (2022)
- Pages: 235-241
- Section: Conference proceedings
- URL: https://journals.rcsi.science/MAJ/article/view/108599
- DOI: https://doi.org/10.17816/MAJ108599
- ID: 108599
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Abstract
BACKGROUND: In the context of the constant manifestation of new SARS-CoV-2 strains and the need to determine the immunogenicity of new variants of antiviral vaccines, it is necessary to create diagnostic test systems based on conservative viral proteins. The SARS-CoV-2 nucleocapsid protein can be considered as a candidate antigen. However, the relevance of existing test systems based on it for determining the titer of antibodies produced in response to infection by recently emerging strains is unknown.
AIM: The goal is to optimize the conditions for obtaining recombinant N proteins of various SARS-CoV-2 strains and to analyze the possibility of creating ELISA test systems based on them.
MATERIALS AND METHODS: Bacterial strains producing N proteins were obtained by amplifying the corresponding genes and ligating them into the pETDuet-1 expression vector. Expression was induced at 20 or 37°C for 1, 2, 4, or 20 h using inducer (IPTG) concentrations of 0.1 mM or 0.5 mM with or without the addition of 3% ethanol. Proteins were purified from biomass by metal affinity chromatography and used as antigens for the detection of antiviral antibodies by ELISA.
RESULTS: It was found that the concentration of the inductor sufficient for the expression of recombinant proteins is 0.1 mM, the induction time is 1 h, and the required temperature is 37 °C. The influence of the presence of ethanol as an expression-stimulating reagent was not revealed. When determining the titers of antiviral antibodies using the obtained proteins, cross-reactivity of serums of COVID-19 convalescents was established regarding to antigens of various SARS-CoV-2 strains.
CONCLUSIONS: The possibility of effective induction of protein synthesis at a minimum concentration of the inducer and cultivation time indicates the economy of its production, and antigen recognition by antiviral antibodies indicates a native structure. Cross-reactivity of the blood sera of convalescents indicates the slow character of the evolution of the antigenic properties of the SARS-CoV-2 N protein. Thus, the purified proteins can be used as a basis for development of diagnostic test systems.
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##article.viewOnOriginalSite##About the authors
Alexandra Ya. Rak
Institute of Experimental Medicine
Author for correspondence.
Email: rak.ay@iemspb.ru
ORCID iD: 0000-0001-5552-9874
Cand. Sci. (Biol.), Senior Research Associate of Laboratory of Immunology and Prophylaxis of Viral Infections of Department of Virology
Russian Federation, Saint PetersburgSvetlana A. Donina
Institute of Experimental Medicine
Email: sveta.donina@gmail.com
ORCID iD: 0000-0002-6502-8341
Scopus Author ID: 6602276916
MD, Cand. Sci. (Med.), Senior Research Associate of Laboratory of Immunology and Prophylaxis of Viral Infections of Department of Virology
Russian Federation, Saint PetersburgIrina N. Isakova-Sivak
Institute of Experimental Medicine
Email: isakova.sivak@iemspb.ru
ORCID iD: 0000-0002-2801-1508
SPIN-code: 3469-3600
Scopus Author ID: 23973026600
Dr. Sci. (Biol.), Head of Laboratory of Immunology and Prevention of Viral Infections, A.A. Smorodintsev Department of Virology
Russian Federation, Saint PetersburgLarisa G. Rudenko
Institute of Experimental Medicine
Email: vaccine@mail.ru
ORCID iD: 0000-0002-0107-9959
SPIN-code: 4181-1372
Scopus Author ID: 7005033248
MD, Dr. Sci. (Med.), Professor, Head of the A.A. Smorodintsev Department of Virology
Russian Federation, Saint PetersburgReferences
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