中国生物工程杂志

Objective: To express the B subunit of Shiga-like Toxin type Ⅱ, and analyze its expression form and receptor-binding activity. Methods: The slt2b gene was obtained from EHEC O157:H7 by PCR, and cloned to the expression vector pET22b(+).The genetically engineered bacteria pET22b(+)-stx2B/BL21 expressed the recombinant StxB after induced with IPTG. The renatured inclusion bodies were purified by ion exchange chromatography. The expression form of rStx2B was investigated by denaturing and native electrophoresis. The receptor-binding activity was confirmed by fluorescence detection and flow cytometer. Result: The constructed genetically engineered bacteria expressed the rStx2B at a high level. The purified protein was obtained after denaturation, renaturation and ion exchange chromatography. According to the denaturing and native electrophoresis, the rStx2B was expressed in a dimmer form, which consists of two monomers cross linked with disulfide bridge. The rStx2B showed good receptor-binding activity by Hela-binding assay. Conclusion: The genetically engineered bacteria were constructed successfully. The receptor-binding activity of rStx2B was independent of the pentamers.