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Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection
Published on: May 2, 2011
[Preparation and identification of rat polyclonal antibody against SARS-CoV-2 main protease (Mpro)]
Gangan Yan1, Haiyan Qi1, Haohao Yan1
1Institute for Drug Screening and Evaluation, Wannan Medical College, Wuhu 241002, China.
Abstract:
Objective To investigate the immunological functions of SARS-CoV-2 main protease (Mpro) in coronavirus disease 2019 (COVID-19), polyclonal antibody against Mpro was developed. Methods A codon-optimized SARS-CoV-2 Mpro gene was synthesized and ligated into a pET-28a vector for construction of a recombinant plasmid named by pET-28a-Mpro. Subsequently, this plasmid was transformed into E.coli Rosetta (DE3) competent cells for Mpro expression in an optimized condition, and then Mpro was purified using a HisTrap chelating column. The purified Mpro was used as immunogen to inoculate rats and the serum was collected after third immunization cycle. The titer, selectivity and sensitivity of polyclonal antibody against Mpro were analyzed using the ELISA and Western blot analysis. Results An optimized expression condition in E.coli cells for Mpro was determined, and the recombinant Mpro was purified by a HisTrap chelating column. The ELISA and Western blot analysis demonstrated that the highly sensitive polyclonal antibody against Mpro specially recognized the recombinant Mpro, and the titer reached 1:256 000. Conclusion The highly specific polyclonal antibody against SARS-CoV-2 Mpro is successfully prepared, which lays an experimental foundation for investigating the immunological function of Mpro in COVID-19.
Insights
Researchers developed a highly sensitive polyclonal antibody against the SARS-CoV-2 main protease (Mpro). This antibody specifically recognizes Mpro, paving the way for further investigation into its immunological functions in COVID-19.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- The SARS-CoV-2 main protease (Mpro) is crucial for viral replication.
- Understanding the immunological functions of Mpro is essential for developing effective COVID-19 therapies.
Purpose of the Study:
- To develop a specific polyclonal antibody against SARS-CoV-2 Mpro.
- To investigate the immunological roles of Mpro in COVID-19 pathogenesis.
Main Methods:
- Synthesized and cloned the SARS-CoV-2 Mpro gene into a pET-28a vector.
- Expressed and purified recombinant Mpro in E. coli.
- Generated and characterized polyclonal antibodies against Mpro using ELISA and Western blot.
Main Results:
- Optimized Mpro expression and purification protocols.
- Developed a highly sensitive and specific polyclonal antibody against Mpro.
- Achieved a high antibody titer of 1:256,000, demonstrating strong Mpro recognition.
Conclusions:
- Successfully prepared a highly specific polyclonal antibody against SARS-CoV-2 Mpro.
- The developed antibody provides a valuable tool for studying Mpro's immunological functions.
- This research lays the groundwork for further exploration of Mpro's role in COVID-19.
