Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2

authors:

avatar Hossein Samiei Abianeh , avatar Shahram Nazarian ORCID , * , avatar Jafar Amani ORCID , avatar Emad Kordbache , avatar Amir Rezaie

Koomesh: Vol.24, issue 6; 727-735
published online: August 02, 2022
article type: Research Article
received: February 09, 2022
accepted: June 02, 2022

how to cite: Samiei Abianeh H, Nazarian S, Amani J, Kordbache E, Rezaie A. Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2. koomesh. 2022;24(6):e152785. 

Abstract

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Materials and Methods: B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Results: The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high- frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion: According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.

References

  • 1.

    Shimizu K. 2019-nCoV, fake news, and racism. Lancet 2020; 395: 685-686.##https://doi.org/10.1016/S0140-6736(20)30357-3.

  • 2.

    Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020; 395: 497-506.##https://doi.org/10.1016/S0140-6736(20)30183-5.

  • 3.

    Dahlke C, Heidepriem J, Kobbe R, Santer R, Koch T, Fathi A, et al. Distinct early IgA profile may determine severity of COVID-19 symptoms: an immunological case series. Med Rxiv 2020.##https://doi.org/10.1101/2020.04.14.20059733.

  • 4.

    Harrison AG, Lin T, Wang P. Mechanisms of SARS-CoV-2 transmission and pathogenesis. Trends Immunol 2020; 41: 1100-1115.##https://doi.org/10.1016/j.it.2020.10.004.

  • 5.

    Wu F, Zhao S, Yu B, Chen Y-M, Wang W, Song Z-G, et al. A new coronavirus associated with human respiratory disease in China. Nature 2020; 579: 265-269.##https://doi.org/10.1038/s41586-020-2008-3.

  • 6.

    Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 2020; 395: 565-574.##https://doi.org/10.1016/S0140-6736(20)30251-8.

  • 7.

    Cheng Y, Feng Y, Luo P, Gu J, Yu S, Zhang WJ, et al. Fusion expression and immunogenicity of EHEC EspA-Stx2Al protein: implications for the vaccine development. J Microbiol 2009; 47: 498-505.##https://doi.org/10.1007/s12275-009-0116-8.

  • 8.

    Tilocca B, Soggiu A, Musella V, Britti D, Sanguinetti M, Urbani A, et al. Molecular basis of COVID-19 relationships in different species: a one health perspective. Microbes Infect 2020; 22: 218-220.##https://doi.org/10.1016/j.micinf.2020.03.002.

  • 9.

    Meyer B, Drosten C, Mller MA. Serological assays for emerging coronaviruses: challenges and pitfalls. Virus Res 2014; 194: 175-183.##https://doi.org/10.1016/j.virusres.2014.03.018.

  • 10.

    Tay MZ, Poh CM, Rnia L, MacAry PA, Ng LF. The trinity of 467 COVID-19: immunity, inflammation and intervention. Nat Rev Immunol 2020; 20: 363-374.

  • 11.

    Mercurio I, Tragni V, Busto F, De Grassi A, Pierri CL. Protein structure analysis of the interactions between SARS-CoV-2 spike protein and the human ACE2 receptor: from conformational changes to novel neutralizing antibodies. Cell Mol Life Sci 2021; 78: 1501-1522.##https://doi.org/10.1007/s00018-020-03580-1.

  • 12.

    Zhang J, Xie B, Hashimoto K. Current status of potential therapeutic candidates for the COVID-19 crisis. Brain Behav Immun 2020; 87: 59-73.##https://doi.org/10.1016/j.bbi.2020.04.046.

  • 13.

    Cuesta-Herranz J, de las Heras M, Fernndez M, Lluch M, Figueredo E, Umpierrez A, et al. Allergic reaction caused by local anesthetic agents belonging to the amide group. J Allergy Clin Immunol 1997; 99: 427-428.##https://doi.org/10.1016/S0091-6749(97)70064-2.

  • 14.

    He Y, Zhou Y, Wu H, Kou Z, Liu S, Jiang S. Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus. J Clin Microbiol 2004; 42: 5309-5314.##https://doi.org/10.1128/JCM.42.11.5309-5314.2004.

  • 15.

    Masters PS, Sturman LS. Background paper functions of the coronavirus nucleocapsid protein. Adv Exp Med Biol 1990; 276: 235-238.##https://doi.org/10.1007/978-1-4684-5823-7_32.

  • 16.

    Kopecky-Bromberg SA, Martnez-Sobrido L, Frieman M, Baric RA, Palese P. Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists. J Virol 2007; 81: 548-557.##https://doi.org/10.1128/JVI.01782-06.

  • 17.

    Surjit M, Liu B, Chow VT, Lal SK. The nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks S phase progression in mammalian cells. J Biol Chem 2006; 281: 10669-10681.##https://doi.org/10.1074/jbc.M509233200.

  • 18.

    Zhu Y, Liu M, Zhao W, Zhang J, Zhang X, Wang K, et al. Isolation of virus from a SARS patient and genome-wide analysis of genetic mutations related to pathogenesis and epidemiology from 47 SARS-CoV isolates. Virus Genes 2005; 30: 93-102.##https://doi.org/10.1007/s11262-004-4586-9.

  • 19.

    Hotez PJ, Corry DB, Strych U, Bottazzi ME. COVID-19 vaccines: neutralizing antibodies and the alum advantage. Nat Rev Immunol 2020; 20: 399-400.##https://doi.org/10.1038/s41577-020-0358-6.

  • 20.

    Wang H, Zhang Q, Shishido T, Takehira KJ. Heteroscedasticity and non-monotonic efficiency effects of a stochastic frontiermodel. J Produc Anal 2002; 18: 241-253.##https://doi.org/10.1023/A:1020638827640.

  • 21.

    Wang C, Li W, Drabek D, Okba N, van Haperen R, Osterhaus AD, et al. A human monoclonal antibody blocking SARS-CoV-2 infection. Nat Commun 2020; 11: 1-6.##https://doi.org/10.1038/s41467-020-16256-y.

  • 22.

    Long QX, Tang XJ, Shi QL, Li Q, Deng HJ, Yuan J, et al. Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nat Med 2020; 26: 1200-1204.##https://doi.org/10.1038/s41591-020-0965-6.

  • 23.

    Okba NM, Mller MA, Li W, Wang C, GeurtsvanKessel CH, Corman VM, et al. Severe acute respiratory syndrome coronavirus 2 specific antibody responses in coronavirus disease patients. Emerging Infect Dis 2020; 26: 1478.##https://doi.org/10.3201/eid2607.200841.

  • 24.

    Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, et al. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 2019; 47: W636-W641.##https://doi.org/10.1093/nar/gkz268.

  • 25.

    Saha S, Raghava GP. Prediction of continuous Bcell epitopes in an antigen using recurrent neural network. Proteins 2006; 65: 40-48.##https://doi.org/10.1002/prot.21078.

  • 26.

    Wang Z, Schmidt F, Weisblum Y, Muecksch F, Barnes CO, Finkin S, et al. mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. Nature 2021; 592: 616-622.##https://doi.org/10.1038/s41586-021-03324-6.

  • 27.

    Dagan N, Barda N, Kepten E, Miron O, Perchik S, Katz MA, et al. BNT162b2 mRNA Covid-19 vaccine in a nationwide mass vaccination setting. N Engl J Med 2021; 384: 1412-1423.##https://doi.org/10.1056/NEJMoa2101765.

  • 28.

    Keehner J, Horton LE, Pfeffer MA, Longhurst CA, Schooley RT, Currier JS, et al. SARS-CoV-2 infection after vaccination in health care workers in California. N Engl J Med 2021; 384: 1774-1775.##https://doi.org/10.1056/NEJMc2101927.

  • 29.

    Oliveira SC, de Magalhes MT, Homan EJ. Immunoinformatic analysis of SARS-CoV-2 nucleocapsid protein and identification of COVID-19 vaccine targets. Front Immunol 2020; 2758.##https://doi.org/10.3389/fimmu.2020.587615.

  • 30.

    Mierendorf RC, Morris BB, Hammer B, Novy RE. Expression and purification of recombinant proteins using the pET system. Methods Mol Med 1998; 13: 257-292.##https://doi.org/10.1385/0-89603-485-2:257.

  • 31.

    Djukic T, Mladenovic M, Stanic-Vucinic D, Radosavljevic J, Smiljanic K, Sabljic L, et al. Expression, purification and immunological characterization of recombinant nucleocapsid protein fragment from SARS-CoV-2. Virology 2021; 557: 15-22.##https://doi.org/10.1016/j.virol.2021.01.004.

  • 32.

    Leenaars M, Hendriksen CF. Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. ILAR J 2005; 46: 269-279.##https://doi.org/10.1093/ilar.46.3.269.

  • 33.

    Shi J, Zhang J, Li S, Sun J, Teng Y, Wu M, et al. Epitope-based vaccine target screening against highly pathogenic MERS-CoV: an in silico approach applied to emerging infectious diseases. PloS One 2015; 10: e0144475.##https://doi.org/10.1371/journal.pone.0144475.

  • 34.

    Veit S, Jany S, Fux R, Sutter G, Volz A. CD8+ T cells responding to the Middle East respiratory syndrome coronavirus nucleocapsid protein delivered by vaccinia virus MVA in mice. Viruses 2018; 10: 718.##https://doi.org/10.3390/v10120718.

  • 35.

    Le Bert N, Tan AT, Kunasegaran K, Tham CY, Hafezi M, Chia A, et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 2020; 584: 457.##https://doi.org/10.1038/s41586-020-2550-z.