Expression of Receptor Binding Domain (RBD) from Coronavirus Spike Protein Fused to Carboxylic Terminal of Clostridium perfringens Enterotoxin (c-CPE) in Pichia pastoris

How to Cite:Elham BehvandiGhasem BagherpourKeyvan NedaeiSaeid KaboliBehrooz JohariExpression of Receptor Binding Domain (RBD) from Coronavirus Spike Protein Fused to Carboxylic Terminal of Clostridium perfringens Enterotoxin (c-CPE) in Pichia pastoris.koomesh.26(2):e149316.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.

Keywords

Recombinant Fusion Proteins

Cloning

Spike Protein

Pichia pastoris

Fulltext

Full text is available in PDF file.

comments

Crossmark

Checking

Share on

Comments

Number of Comments:0

Cited by

CrossRef 0

Metrics

Get Permission (article level)

Purchasing Reprints

Copyright Clearance Center (CCC) handles bulk orders for article reprints for Brieflands. To place an order for reprints, please click here ( https://www.copyright.com/landing/reprintsinquiryform/ ). Clicking this link will bring you to a CCC request form where you can provide the details of your order. Once complete, please click the ‘Submit Request’ button and CCC’s Reprints Services team will generate a quote for your review.

Search Relations

Author(s):

Elham Behvandi:[PubMed][Scholar]
Ghasem Bagherpour:[PubMed][Scholar]
Keyvan Nedaei:[PubMed][Scholar]
Saeid Kaboli:[PubMed][Scholar]
Behrooz Johari:[PubMed][Scholar]