Optimization of purification conditions and study of antigenic properties of recombinant nucleocapsid protein of different SARS-CoV-2 strains

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.

Keywords

antigen / protein expression / recombinant protein / COVID-19 convalescents / nucleocapsid protein / SARS-CoV-2 / test system / cross-reactivity

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