The Recombinant Expression and Receptor-binding Activity of the B Subunit of Shiga-like Toxin Type Ⅱ / 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.

Synthesis of DNA Encoding Botulinum Neurotoxin Receptor syt II and Its Fusion Expression in E.coli / 中国生物工程杂志

In order to construct a prokaryotic expression vector of human receptor syt II N-fragment and to express recombinant MBP-Syt fusion protein in E.coli and to purify and identify its activity. According to codon preference of E.coli, a DNA fragment encoding human syt II N-fragment was synthesized, and then cloned into prokaryotic vector pMAL-c2x for sequencing. Then the recombinant plasmid pMAL-Syt was introduced into E.coli ER2566 by transformation for expression and the obtained engineered bacteria were induced by IPTG. The fusion protein was purified by amylose resin affinity chromatography and identified by SDS-PAGE and Western blot. The binding activity of the protein was determined by ELISA. It is concluded that MBP-Syt protein is of good binding activity.

Construction and Screening of a Phage Display Library of Repertoire Single Chain Fv Antibody from Mouse Immunized with BoNTB/Hc / 微生物学通报

To produce antibodies capable of neutralizing botulinum neurotoxin type B(BoNT/B),We cloned the carboxy-terminal end of Hc containing the major determinants responsible for specific toxin,induced and purifed.The heavy-chain and kappa light-chain variable region gene repertoire of immunoglobulin were amplified individually from the spleen cell mRNA by RT-PCR and joined as a single-chain Fv(scFv)DNA fragment.These fragment were cloned into the phagemid pCANTAB5E and the phage display library was constructed.Results showed that the high affinity scFv was obtained after 4 rounds of panning,with its DNA sequence conforming to that of mouse antibody.