Expression and Identification of Virus-like Particles Presenting Novel Coronavirus RBD Antigen

doi:10.13523/j.cb.2112054

China Biotechnology

2022, Vol. 42

Issue (5): 117-123 DOI: 10.13523/j.cb.2112054

Expression and Identification of Virus-like Particles Presenting Novel Coronavirus RBD Antigen

LU Hui-shuang,MA Jia-xiu,JIN Jia-pei,ZHANG Jin,LI Ya-lan,CAI Xue-fei^**(

)

Key Laboratory of Molecular Biology of Infectious Diseases of Ministry of Education, Chongqing Medical University,Chongqing 400016,China

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Abstract

Objective: Hepatitis B virus core protein HBc was used as vector to construct virus-like particles expressing novel coronavirus spike protein receptor binding domain RBD, and their immunogenicity was identified, which provides a new idea for the development of COVID-19 vaccines. Methods: The amino acid coding sequence 78 and 81 of hepatitis B virus core protein HBc (1-183 aa) were inserted into novel coronavirus spike protein receptor binding domain RBD and ligated by flexible linker (G4S) 3. After sequence optimization, the fusion gene was cloned into prokaryotic expression vector pET-28a (+) and transformed into expression strain Rosetta. After induced expression in self-inducing medium, the virus-like particles (VLPs) were purified by sucrose density gradient centrifugation. VLPs were detected and identified by SDS-PAGE, Western blot and transmission electron microscope. BALB/c mice were immunized subcutaneously with the prepared VLPs in equal proportion with adjuvant. The specific antibodies in the serum of the mice were analyzed by ELISA to verify the immune effect of HBc-RBD VLPs. Results: Escherichia coli can express partially soluble VLPs in self-inducing medium. VLPs could be observed by transmission electron microscope after purification by sucrose density gradient centrifugation. Mice immunized with HBc-RBD VLPs produced specific antibodies against RBD antigen. Conclusion: VLPs displaying RBD antigen were successfully expressed in prokaryotic expression systems, and their immunogenicity was preliminarily verified by mouse experiment, which provides a new direction for the research and development of novel coronavirus vaccines.

Key words： Virus-like particle(VLP) RBD antigen Hepatitis B core antigen Protein immunogenicity

Received: 24 December 2021 Published: 17 June 2022

ZTFLH:

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Corresponding Authors: Xue-fei CAI E-mail: caixuefei@cqmu.edu.cn

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	Hui-shuang LU
	Jia-xiu MA
	Jia-pei JIN
	Jin ZHANG
	Ya-lan LI
	Xue-fei CAI

Cite this article:

LU Hui-shuang,MA Jia-xiu,JIN Jia-pei,ZHANG Jin,LI Ya-lan,CAI Xue-fei. Expression and Identification of Virus-like Particles Presenting Novel Coronavirus RBD Antigen. China Biotechnology, 2022, 42(5): 117-123.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2112054 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I5/117

Fig.1 Schematic map of HBc-RBD fusion gene

Fig.2 Identification of recombinant plasmid by restriction endonuclease digestion M:DNA marker DL5000 ; 1:Recombinant plasmids; 2: Recombinant plasmids digested by NdeⅠ; 3: Recombinant plasmids digested by NdeⅠand XhoⅠ

Fig.3 Expression of HBc-RBD protein analyzed by SDS-PAGE M:Protein marker; 1: Uninduced bacterial protein; 2: Induced bacterial protein; 3-4: The supernatant and precipitation of bacterial lysis after induction

Fig.4 SDS-PAGE analysis of sucrose density gradient centrifugation M:Protein marker ; 1-12: 12 layer samples of sucrose density gradient centrifugation from top to bottom

Fig.5 SDS-PAGE and Western blot analysis of concentrated protein after dialysis A. SDS-PAGE analysis of concentrated protein after dialysis. M:Protein marker; 1:The 10th layer of sucrose gradient;2:The 9th layer of sucrose gradient;3:The 8th layer of sucrose gradient B. Western blot analysis of concentrated protein after dialysis.(a)Detection of concentrated protein after dialysis by Western blot with anti-HBcAg antibody;(b)Detection of concentrated protein after dialysis by Western blot with anti- RBD antibody

Fig.6 Transmission electron microscope image of virus-like particles

Fig.7 Detection of body weight and specific antibody IgG in serum of immunized mice (a) Body weight changes of immunized mice (b)Detection of anti HBc-RBD VLPs antibody in serum of immunized mice (c) Detection of anti-HBcAg antibody in serum of immunized mice (d)Detection of Anti- RBD antibody in serum of immunized mice


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