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Updated: Aug 25, 2025
Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection
Published on: May 2, 2011
Recombinant expression of SARS-CoV-2 receptor binding domain (RBD) in Escherichia coli and its immunogenicity in mice
Zahra Rahbar1, Shahram Nazarian2, Ruhollah Dorostkar3
1Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Objectives:
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), giving rise to the coronavirus disease 2019 (COVID-19), has become a danger to wellbeing worldwide. Thus, finding efficient and safe vaccines for COVID-19 is of great importance. As a basic step amid contamination, SARS-CoV-2 employs the receptor-binding domain (RBD) of the spike protein to lock in with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells. SARS-CoV-2 receptor-binding domain (RBD) is the main human antibody target for developing vaccines and virus inhibitors, as well as neutralizing antibodies. A bacterial procedure was developed for the expression and purification of the SARS-CoV-2 spike protein receptor-binding domain.
Materials And Methods:
In this research study, RBD was expressed by Escherichia coli and purified with Ni-NTA chromatography. Then it was affirmed by the western blot test. The immunogenicity and protective efficacy of RBD recombinant protein were assessed on BALB/c mice. Additionally, RBD recombinant protein was tested by ELISA utilizing sera of COVID-19 healing patients contaminated with SARS-CoV-2 wild type and Delta variation.
Results:
Indirect ELISA was able to detect the protein RBD in serum of the immunized mouse expressed in E. coli. The inactive SARS-CoV2 was detected by antibodies within the serum of immunized mice. Serum antibodies from individuals recovered from Covid19 reacted to the expressed protein.
Conclusion:
Our findings showed that RBD is of great importance in vaccine design and it can be used to develop recombinant vaccines through induction of antibodies against RBD.
Insights
This study developed a bacterial method to produce the SARS-CoV-2 receptor-binding domain (RBD) for COVID-19 vaccine development. The recombinant RBD protein successfully induced antibodies in mice and reacted with antibodies from recovered COVID-19 patients.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19, a global health threat.
- The SARS-CoV-2 spike protein's receptor-binding domain (RBD) is crucial for host cell entry and a primary target for vaccines and therapeutics.
- Developing effective and safe COVID-19 vaccines is a high priority.
Purpose of the Study:
- To develop a bacterial expression and purification procedure for the SARS-CoV-2 RBD.
- To assess the immunogenicity and protective efficacy of the recombinant RBD protein.
- To evaluate the reactivity of the recombinant RBD with antibodies from COVID-19 patients.
Main Methods:
- Recombinant SARS-CoV-2 RBD was expressed in *Escherichia coli* and purified using Ni-NTA chromatography.
- Protein expression was confirmed by western blot.
- Immunogenicity was assessed in BALB/c mice, and antibody responses were measured by ELISA.
- Reactivity with sera from recovered COVID-19 patients was also tested via ELISA.
Main Results:
- The bacterial expression system successfully produced and purified SARS-CoV-2 RBD.
- Indirect ELISA confirmed the presence of RBD protein in immunized mouse serum.
- Antibodies from immunized mice recognized inactive SARS-CoV-2, and serum from recovered COVID-19 patients reacted with the expressed RBD.
- The recombinant RBD protein demonstrated immunogenicity in mice.
Conclusions:
- The receptor-binding domain (RBD) of SARS-CoV-2 is critical for vaccine design.
- Recombinant vaccines targeting RBD can be developed to induce protective antibodies against SARS-CoV-2.
- Bacterial expression provides a viable method for producing SARS-CoV-2 RBD for vaccine research.
