Preparation of recombinant SARS-CoV-2 nucleocapsid protein

Introduction . The new coronavirus infection (COVID-19) agent SARS-CoV-2 has become widespread in the world and has caused the pandemic that started in 2019. The virus is a zooanthroponotic infectious agent that causes infection in humans as well as in many mammal species. To date, SARS-CoV-2 has been reported both in domestic and in wild animals. Moreover, successful experimental infection of certain animal species was reported during the studies. There is also the evidence that infected animals can transmit the virus to other animals in natural settings through contactincluding virus transmission between animals of different species. Currently, some researchers fear that SARS-CoV-2 may spread to mammalian species in the wild that will become a natural reservoir responsible for this infection outbreaks in humans. Furthermore, the virus effect on potentially susceptible wild animal species, including endangered animal species, is currently not fully understood. Therefore, the infection spread in wild animals requires further study. This requires highly sensitive and specific diagnostic methods. Enzyme-linked immunosorbent assay (ELISA) using SARS-CoV-2 nucleocapsid protein as an antigen can be used for serological surveillance of the new coronavirus infection in animals. Recombinant protein used as an antigen is the most preferable because of its safety. Objective . The study was aimed at preparing highly concentrated recombinant SARS-CoV-2 nucleocapsid protein and testing it for antigenic activity and specificity. Materials and methods . The following was used for the study: SARS-CoV-2, pQE plasmid, Escherichia coli JM109 strain. The following was performed: reverse transcription and polymerase chain reaction, molecular cloning, recombinant protein synthesis, recombinant protein purification, indirect ELISA was used. Results . Molecular cloning of SARS-CoV-2 N-gene was carried out using prokaryotic expression system. Escherichia coli clones producing 33 kDa recombinant SARSCoV-2 nucleocapsid protein were prepared. Optimal expression and purification conditions for highly concentrated antigen preparation were determined. It was shown that optimal inducer concentration was 0.5 mМ, optimal expression period was 4 hours. Urea at a concentration of 8 M as a denaturing agent and optimal imidazole concentration of 0.4 M in the elution buffer were selected based on the results of study of optimal conditions for recombinant antigen purification. Use of the optimal expression and purification procedure allowed us to prepare 1.5 mg of purified antigen from 100 mL of Escherichia coli culture. The recombinant protein demonstrated its high antigenic activity and specificity when tested with indirect ELISA. Conclusion . Preparation of highly concentrated recombinant SARS-CoV-2 nucleocapsid protein enables its further use as an antigen for ELISA test system for detection of antibodies against SARS-CoV-2 nucleocapsid protein in animal sera.