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Background: Vibrio cholera toxin B (CTB) subunit is the pentameric non-toxic portion of cholera toxin (CT) which is responsible for the holotoxin binding to the GM1 ganglioside receptor present on nucleated cells. this study was to produce, purify, and verify recombinant CTB (rCTB) subunitin prokaryotic system. Materials and Methods: In this experimental study, rCTB expression vector (pET-28a) which could be induced in E. coli (BL21) was designed and synthesized. Then the recombinant expression strains containing the result of IPTG interaction were induced and the rCTB was generated on small and large scales. The rCTB produced through Ni2+-charged resin, after refolding and free of possible CTA contaminants, was extracted. After purification, rCTB was verified by Western blotting. Results: The results indicated the level of purification to be about 480µg of purified active pentameric rCTB for each liter of the induced culture. Also, Western blotting analysis showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. Conclusion: The findings of this study demonstrated that E.coli is an available host for production of CTB. In addition, the designed host and vector can be used in large scale production of this protein

Keywords: Cholera Toxin, Purifying, Rctb, Western Blotting

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