Production and Purification of Recombinant B Subunit of Vibrio cholerae Toxin in Escherichia coli

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-۲۲a plasmid in Escherichia coli BL۲۱(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-۲۲a plasmid by NcoI and SacI restriction enzymes and transformed into E. coli. Transformed bacteria were inoculated into a ۲×YT medium. Stimulation of recombinant protein production was performed by adding IPTG with a final concentration of ۰.۴ mM. Finally, recombinant protein was purified by a Ni-IDA column. The concentration of recombinant protein was determined by GM۱-ELISA and Bradford tests. The Western blotting technique verified recombinant CTB, So results showed the expected bands at a molecular weight of about ۱۲.۷۶ kDa (denatured) and ۶۳ kDa (non-denatured). GM۱-ELISA and Bradford tests showed the final protein concentrations of ۱۱ and ۹.۵۷ mg/L, respectively. GM۱-ELISA confirmed the biological activity of rCTB in the presence of GM۱ 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.

کلیدواژه ها:

Cholera toxin B subunit ، Escherichia coli ، Molecular cloning ، Vibrio cholerae