SARS-CoV-2 N-Ferritin原核表达质粒的构建及鉴定

doi:10.3969/j.issn.1001-8131.2025.04.001

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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.

Key words： SARS-CoV-2 Ferritin nanoparticle vaccine Protein purification

Received: 19 May 2025

PACS:

R734.2

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	Articles by authors
	Zeng Youqin
	Wang Qiang
	Yang Kaiwen
	Song Yujie
	Huang Lianjie

Cite this article:

Zeng Youqin,Wang Qiang,Yang Kaiwen, et al. Construction and Characterization of SARS-CoV-2 N-Ferritin Prokaryotic Expression Plasmids[J]. journal1, 2025, 45(4): 1-5.

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http://www.hrbyybjb.org.cn/EN/10.3969/j.issn.1001-8131.2025.04.001 OR http://www.hrbyybjb.org.cn/EN/Y2025/V45/I4/1

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