Construction and Characterization of SARS-CoV-2 N-Ferritin Prokaryotic Expression Plasmids
Zeng Youqin1, Wang Qiang2, Yang Kaiwen1, Song Yujie2, Huang Lianjie1
1. College of Medical Technology,Chengdu University of Traditional Chinese Medicine,Chengdu,611130,China; 2. Sichuan Academy of Medical Sciences Sichuan Provincial People's Hospital (SAMSPH)Chengdu,610072,China
Abstract：Objective To construct the prokaryotic expression vector pET32a-N-Ferritin-Flag-6His by a novel ferritin vaccine platform,optimize the induction expression condition of the recombinant protein N-Ferritin,and characterize its morphological structure and stability in depth. Methods The full-length sequence of the SARS-CoV-2 N(MT108784.1)protein was connected to the N-terminus of the ferritin protein by a flexible linker(G4S)3,and the C-terminus was connected with the Flag and 6×His tags,which was inserted into the pET32a vector,and the recombinant plasmid pET32a-N-Ferritin-Flag-6His of the fusion expression protein N-Ferritin was constructed.The pET32a-N-Ferritin-Flag-6His plasmid was transformed into the BL21(DE3)competent cells,and the optimal induction condition of the recombinant protein N-Ferritin was determined by optimizing the final concentration of IPTG in the induction process,time and temperature.The induced recombinant protein N-Ferritin was purified by nickel affinity chromatography column,and the recombinant protein was verified and identified by SDS-PAGE,Western blot,transmission electron microscopy,and DLS.The stability of the recombinant protein was analyzed by SDS-PAGE and Coomassie brilliant blue staining under different freezing and thawing times and different storage temperatures. Results The recombinant N-Ferritin protein was successfully prepared,and there was a target band at 85 kDa.Transmission electron microscopy observed that the N-Ferritin recombinant protein showed a uniform spherical particle.The DLS results showed that the particle size of the recombinant protein was 18.23 nm.Repeated freezing and thawing experiments showed that the N-Ferritin recombinant protein still had good stability after repeated freezing and thawing 3-5 times and stored for 4 weeks under different temperatures(80℃,20℃,4℃). Conclusion The N-Ferritin prokaryotic expression vector was successfully constructed,and a large amount of high-purity target protein could be obtained after optimizing the induction conditions,and it has good stability.
[1] Lu R,Zhao X,Li J,et al.Genomic characterisation and epidemiology of 2019 novel coronavirus:implications for virus origins and receptor binding[J].Lancet,2020,395(10224):565-74. [2] Harrison A G,Lin T,Wang P.Mechanisms of SARS-CoV-2 Transmission and Pathogenesis[J].Trends Immunol,2020,41(12):1100-15. [3] Wu F,Zhao S,Yu B,et al.A new coronavirus associated with human respiratory disease in China[J].Nature,2020,579(7798):265-9. [4] He Y,Zhou Y,Wu H,et al.Mapping of antigenic sites on the nucleocapsid protein of the severe acute respiratory syndrome coronavirus[J].J Clin Microbiol,2004,42(11):5309-14. [5] Masters P S,Sturman L S.Background paper.Functions of the coronavirus nucleocapsid protein[J].Adv Exp Med Biol,1990,276:235-8. [6] Kopecky-Bromberg S A,Martínez-Sobrido L,Frieman M,et al.Severe acute respiratory syndrome coronavirus open reading frame(ORF)3b,ORF 6,and nucleocapsid proteins function as interferon antagonists[J].J Virol,2007,81(2):548-57. [7] Surjit M,Liu B,Chow V T,et al.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].J Biol Chem,2006,281(16):10669-81. [8] Li Y H,Li J,Liu X E,et al.Detection of the nucleocapsid protein of severe acute respiratory syndrome coronavirus in serum:comparison with results of other viral markers[J].J Virol Methods,2005,130(1-2):45-50. [9] Surjit M,Lal S K.The SARS-CoV nucleocapsid protein:a protein with multifarious activities[J].Infect Genet Evol,2008,8(4):397-405. [10] Ahlén G,Frelin L,Nikouyan N,et al.The SARS-CoV-2 N Protein Is a Good Component in a Vaccine[J].J Virol,2020,94(18). [11] Dutta N K,Mazumdar K,Gordy J T.The Nucleocapsid Protein of SARS-CoV-2:a Target for Vaccine Develop-ment[J].J Virol,2020,94(13). [12] Li D,Song H,Li J,et al.Expression and Evaluation of a Novel PPRV Nanoparticle Antigen Based on Ferritin Self-Assembling Technology[J].Pharmaceutics,2022,14(9). [13] Zhao Z,Chen X,Chen Y,et al.A Self-Assembling Ferritin Nanoplatform for Designing Classical Swine Fever Vac-cine:Elicitation of Potent Neutralizing Antibody[J].Vac-cines(Basel),2021,9(1). [14] Zhu Y,Zhu Y,Cao T,et al.Ferritin-based nanomedicine for disease treatment[J].Med Rev(2021),2023,3(1):49-74. [15] Cong Y,Ulasli M,Schepers H,et al.Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle[J].J Virol,2020,94(4). [16] Grifoni A,Sidney J,Zhang Y,et al.A Sequence Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to SARS-CoV-2[J].Cell Host Microbe,2020,27(4):671-80.e2. [17] Marra M A,Jones S J,Astell C R,et al.The Genome se-quence of the SARS-associated coronavirus[J].Science,2003,300(5624):1399-404. [18] Zhou R,To K K,Wong Y C,et al.Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses[J].Immunity,2020,53(4):864-77.e5. [19] Oliveira S C,De Magalh es M T Q,Homan E J.Im-munoinformatic Analysis of SARS-CoV-2 Nucleoca-psid Protein and Identification of COVID-19 Vaccine Targets[J].Front Immunol,2020,11:587615. [20] Le Bert N,Tan A T,Kunasegaran K,et al.SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS,and uninfected controls[J].Nature,2020,584(7821):457-62. [21] Ng O W,Chia A,Tan A T,et al.Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection[J].Vaccine,2016,34(17):2008-14. [22] Grifoni A,Weiskopf D,Ramirez S I,et al.Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals[J].Cell,2020,181(7):1489-501.e15. [23] Gao W,Tamin A,Soloff A,et al.Effects of a SARS-associated coronavirus vaccine in monkeys[J].Lancet,2003,362(9399):1895-6. [24] Okada M,Takemoto Y,Okuno Y,et al.The development of vaccines against SARS corona virus in mice and SCID-PBL/hu mice[J].Vaccine,2005,23(17-18):2269-72.
2025, Vol. 45 
