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.

1. On-line coronavirus COVID-19 map. https://coronavirus-monitor.info (in Russ.)

2. Mahdy M. A. A., Younis W., Ewaida Z. An overview of SARS-CoV-2 and animal infection. Frontiersin Veterinary Science. 2020; 7:596391. https://doi.org/10.3389/fvets.2020.596391

3. Enserink M. Coronavirus rips through Dutch mink farms, triggering culls. Science. 2020; 368 (6496):1169. https://doi.org/10.1126/science.368.6496.1169

4. McAloose D., Laverac M., Wang L., Killian M. L., Caserta L. C., YuanF., et al. Frompeople toPanthera: Natural SARS-CoV-2 infectionintigers and lions at the Bronx Zoo. mBio. 2020; 11 (5):e02220-20. https://doi.org/10.1128/mbio.02220-20

5. Oreshkova N., Molenaar R. J., Vreman S., Harders F., Oude Munnink B. B., Hakze-van der Honing R. W., et al. SARS-CoV-2 infection in farmed minks, the Netherlands, April and May 2020. Eurosurveillance. 2020; 25 (23):2001005. https://doi.org/10.2807/1560-7917.es.2020.25.23.2001005

6. Sailleau C., Dumarest M., Vanhomwegen J., Delaplace M., Caro V., Kwasiborski A., et al. First detection and genome sequencing of SARS-CoV-2 in an infected cat in France. Transboundary and Emerging Diseases. 2020; 67 (6): 2324–2328. https://doi.org/10.1111/tbed.13659

7. Freuling C. M., Breithaupt A., Müller T., Sehl J., Balkema-Buschmann A., Rissmann M., et al. Susceptibility of raccoon dogs for experimental SARS-CoV-2 infection. Emerging Infectious Diseases. 2020; 26 (12): 2982–2985. https://doi.org/10.3201/eid2612.203733

8. Sit T. H. C., Brackman C. J., Ip S. M., Tam K. W. S., Law P. Y. T., To E. M. W., et al. Infection of dogs with SARS-CoV-2. Nature. 2020; 586: 776–778. https://doi.org/10.1038/s41586-020-2334-5

9. Schlottau K., Rissmann M., Graaf A., Schön J., SehlJ., Wylezich C., et al. SARS-CoV-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study. The Lancet Microbe. 2020; 1 (5): e218–e225. https://doi.org/10.1016/s2666-5247(20)30089-6

10. Hobbs E. С., Reid T. J. Animals and SARS-CoV-2: Species susceptibility and viral transmission in experimental and natural conditions, and the potential implications for community transmission. Transboundary and Emerging Diseases. 2021; 68 (4): 1850–1867. https://doi.org/10.1111/tbed.13885

11. Makeeva Yu. Thirty-five WOAH member countries reported SARSCoV-2 in animals. Veterinary Medicine and Life. December 28, 2022. https:// vetandlife.ru/sobytiya/o-vyyavlenii-sars-cov-2-u-zhivotnyh-zayaviliv-vozzh-35-stran (in Russ.)

12. EFSA Panel on Animal Health and Welfare (AHAW), Nielsen S. S., Alvarez J., Bicout D. J., Calistri P., Canali E., et al. SARS-CoV-2 in animals: susceptibility of animal species, risk for animal and public health, monitoring, prevention and control. EFSA Journal. 2023; 21 (2):e07822. https://doi.org/10.2903/j.efsa.2023.7822

13. Fang R., Yang X., Guo Y., Peng B., Dong R., Li S., Xu S. SARS-CoV-2 infection in animals: Patterns, transmission routes, and drivers. EcoEnvironment & Health. 2024; 3 (1): 45–54. https://doi.org/10.1016/j.eehl.2023.09.004

14. Mastutik G., Rohman A., I’tishom R., Ruiz-Arrondo I., de BlasI. Experimental and natural infections ofsevere acute respiratory syndrome-related coronavirus 2 in pets and wild and farm animals. Veterinary World. 2022; 15 (3): 565–589. https://doi.org/10.14202/vetworld.2022.565-589

15. Cui S., LiuY., Zhao J., Peng X., LuG., ShiW., et al. An updated review on SARS-CoV-2 infection in animals. Viruses. 2022; 14 (7):1527. https://doi.org/10.3390/v14071527

16. Hammer A. S., Quaade M. L., Rasmussen T. B., Fonager J., Rasmussen M., Mundbjerg K., et al. SARS-CoV-2 transmission between mink (Neovison vison) and humans, Denmark. Emerging InfectiousDiseases. 2021; 27 (2): 547–551. https://doi.org/10.3201/eid2702.203794

17. Yen H.-L., Sit T. H. C., Brackman C. J., Chuk S. S. Y., Gu H., Tam K. W. S., et al. Transmission of SARS-CoV-2 delta variant (AY.127) from pet hamsters to humans, leading to onward human-to-human transmission: a case study. The Lancet. 2022; 399 (10329): 1070–1078. https://doi.org/10.1016/s0140-6736(22)00326-9

18. Cui X., Wang Y., Zhai J., Xue M., Zheng C., Yu L. Future trajectory of SARS-CoV-2: Constant spillover back and forth between humans and animals. Virus Research. 2023; 328:199075. https://doi.org/10.1016/j.virusres.2023.199075

19. Cheng K., Wu C., Gu S., LuY., Wu H., Li C. WHOdeclaresthe end of the COVID-19 global health emergency: lessons and recommendations from the perspective of ChatGPT/GPT-4. International Journal of Surgery. 2023; 109 (9): 2859–2862. https://doi.org/10.1097/js9.0000000000000521

20. Santini J. M., Edwards S. J. L. Host range of SARS-CoV-2 and implications for public health. The Lancet Microbe. 2020; 1 (4): e141–e142. https://doi.org/10.1016/s2666-5247(20)30069-0

21. Fritz M., Elguero E., Becquart P., de Fonclare D. de R., Garcia D., Beurlet S., et al. A large-scale serological survey in pets from October 2020 through June 2021 in France shows significantly higher exposure to SARSCoV-2 in cats. bioRxiv. December 26, 2022; preprint, Version 4. https://doi.org/10.1101/2022.12.23.521567

22. Wang A., Zhu X., Chen Y., Sun Y., Liu H., Ding P., et al. Serological survey of SARS-CoV-2 in companion animalsin China. Frontiersin Veterinary Science. 2022; 9:986619. https://doi.org/10.3389/fvets.2022.986619

23. Goldberg A. R., Langwig K. E., Brown K. L., Marano J. M., Rai P., King K. M., et al. Widespread exposure to SARS-CoV-2 in wildlife communities. Nature Communications. 2024; 15 (1):6210. https://doi.org/10.1038/s41467-024-49891-w.

24. Volkova M. A., Zinyakov N. G., Yaroslavtseva P. S., Chvala I. A., Galkina T. S., Andreychuk D. B. Development of the test kit for detection of SARSCoV-2 antibodies in sera of susceptible animals. Veterinary Science Today. 2021; (2): 97–102. https://doi.org/10.29326/2304-196X-2021-2-37-97-102

25. Burbelo P. D., Riedo F. X., Morishima C., Rawlings S., Smith D., Das S., et al. Sensitivity in detection of antibodies to nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 in patients with coronavirus disease 2019. The Journal of Infectious Diseases. 2020; 222 (2): 206–213. https://doi.org/10.1093/infdis/jiaa273

26. De Marco Verissimo C., O’Brien C., López Corrales J., Dorey A., Cwiklinski K., Lalor R., et al. Improved diagnosis of SARS-CoV-2 by using nucleoprotein and spike protein fragment 2 in quantitative dual ELISA tests. Epidemiology and Infection. 2021; 149:e140. https://doi.org/10.1017/S0950268821001308

27. Gribanov O. G., Shcherbakov A. V., Perevozchikova N. A., Gusev A. A. Purification of DNA fragments, plasmid DNA, and RNA using aerosil A-300 and GF/F (GF/C) filters. Biochemistry (Moscow). 1996; 61 (6): 764–768. https://elibrary.ru/ldmgwz

28. ManiatisT., Fritsch E. F., Sambrook J. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory; 1982. 545 p.