Ferdowsi University of Mashhad

Document Type : Research Articles

Authors

1 Department of Biotechnology Research, Mashhad Branch, Razi Vaccine and Serum Research Institute, Agriculture Research, Education and Extension Organization (AREEO), Mashhad, Iran

2 Department of Computer Engineering, Imam Reza International University, Mashhad, Iran

Abstract

Cholera toxin B subunit (CTB) is a non-toxic and immunostimulatory component of Vibrio cholera toxin. CTB is one of the most studied protein compounds for adjuvant design. This study aimed to produce and purify recombinant CTB (rCTB) by pET-22a plasmid in Escherichia coli BL21(DE) strain, focusing on cost-effectiveness and ease of use. The target gene was identified in the genome of Vibrio cholerae biotype El Tor through the NCBI database, and its specific primers were designed. The gene fragment was amplified by PCR and cloned into pET-22a plasmid by NcoI and SacI restriction enzymes and transformed into E. coli. Transformed bacteria were inoculated into a 2×YT medium. Stimulation of recombinant protein production was performed by adding IPTG with a final concentration of 0.4 mM. Finally, recombinant protein was purified by a Ni-IDA column. The concentration of recombinant protein was determined by GM1-ELISA and Bradford tests. The Western blotting technique verified recombinant CTB, So results showed the expected bands at a molecular weight of about 12.76 kDa (denatured) and 63 kDa (non-denatured). GM1-ELISA and Bradford tests showed the final protein concentrations of 11 and 9.57 mg/L, respectively. GM1-ELISA confirmed the biological activity of rCTB in the presence of GM1 ganglioside receptor. Recombinant CTB produced by the method proposed in this research has high purity and appropriate concentration and can be used in immunological studies, especially adjuvant design.

Keywords

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