Prokaryotic expression, purification and immunogenicity of SARS-CoV-2 omicron variant nucleocapsid protein.
- Author:
Zewen TU
1
;
Quansheng WANG
1
;
Shiguo LIU
1
;
Haosen LIU
2
;
Chunyan ZENG
2
;
Juanjuan XIE
2
;
Mingzhi LI
1
;
Jingcai LI
2
;
Min WANG
2
;
Shiqi WENG
3
;
Lumei KANG
4
;
Lingbao KONG
5
Author Information
1. College of Biological Science and Engineering, Jiangxi Agricultural University, Nanchang 330045; Nanchang Key Laboratory of Animal Virus and Genetic Engineering, Nanchang 330045, China.
2. College of Biological Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China.
3. Department of Clinical Laboratory, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, China.
4. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045; Center of Laboratory Animal Science and Technology, Nanchang University, Nanchang 330031, China.
5. College of Biological Science and Engineering, Jiangxi Agricultural University, Nanchang 330045; Nanchang Key Laboratory of Animal Virus and Genetic Engineering, Nanchang 330045, China. *Corresponding author, E-mail: lingbaok@hotmail.com.
- Publication Type:Journal Article
- MeSH:
Animals;
Mice;
SARS-CoV-2/genetics*;
Coronavirus Nucleocapsid Proteins/immunology*;
Nucleocapsid Proteins/isolation & purification*;
COVID-19/immunology*;
Antibodies, Viral/immunology*;
Mice, Inbred BALB C;
Interferon-gamma/metabolism*;
Interleukin-1beta/metabolism*;
Female;
Escherichia coli/metabolism*;
Mutation;
Humans
- From:
Chinese Journal of Cellular and Molecular Immunology
2025;41(8):735-743
- CountryChina
- Language:Chinese
-
Abstract:
Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
