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Journal of Semnan University of Medical Sciences
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Expression of Receptor Binding Domain (RBD) from Coronavirus Spike Protein Fused to Carboxylic Terminal of Clostridium perfringens Enterotoxin (c-CPE) in Pichia pastoris
Author(s):
1Department of Medical Biotechnology, Zanjan University of Medical Sciences, Zanjan, Iran
2Zanjan Pharmaceutical Biotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, Iran
*Corresponding Author: Department of Medical Biotechnology, Zanjan University of Medical Sciences, Zanjan, Iran. Email: [email protected]
How to Cite:Behvandi E, Bagherpour G, Nedaei K, Kaboli S, Johari B. Expression of Receptor Binding Domain (RBD) from Coronavirus Spike Protein Fused to Carboxylic Terminal of Clostridium perfringens Enterotoxin (c-CPE) in Pichia pastoris. koomesh. 2024;26(2):e149316. doi: https://doi.org/10.69107/koomesh-149316
Abstract
Background: To enhance mucosal IgA, administering intra-nasal antigen transfer may be considered. One challenge with this approach is antigenic factors' limited penetration of nasal mucosa. This research utilized c-CPE, the carboxylic end-section of the enterotoxin Clostridium perfringens. Specifically, the RBD-c-CPE fusion structure was cloned in a bacterial host and integrated into the yeast host genome for protein expression.
Objectives: The primary aim of this investigation is to develop methods: Following the construction of the expression structure, it was then cloned into the yeast vector a coronavirus vaccine that can elicit sufficient immunity in the host.
Methods: Materials pPICZαA: Subsequently, the TOP10F bacterium was transformed with the recombinant plasmid, and plasmid extraction was carried out. This was followed by the transformation of yeast strain X33 with linearized recombinant plasmids. Positive clones obtained from the overnight sowing process were utilized for the expression stages. To confirm protein secretion in the yeast medium, the protein concentration, western blot, and ECL were performed.
Results: Agarose gel electrophoresis results indicated the successful construction of the RBD-c-CPE gene. Colony PCR analysis of both TOP10F bacteria and Pichia pastoris yeast post-transformation confirmed the effective cloning of the recombinant pPIC-RC plasmid in TOP10F bacteria and the successful integration of the recombinant plasmid into the yeast genome, respectively. Moreover, Western blot analysis provided conclusive evidence of the successful expression of the RBD-c-CPE fusion protein in Pichia pastoris yeast.
Conclusions: The RBD-c-CPE has been effectively integrated and produced within the yeast host genome following its cloning in a bacterial host. Further enhancing the fused protein's expression can serve as a potential candidate for preclinical trials on animals for a COVID-19 mucosal vaccine.
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