The expression of receptor-binding region of Clostridium difficile toxin B

Abstract

Objective To express receptor-binding region of Clostridium difficile toxin B by gene cloning,providing foundation forestablishmentof a rapid method to detect Clostridium difficile. Methods Recover Clostridium difficile,extract genomic DNA and amplify the targeted gene by PCR.The recovered PCR product was linked to p GEM-T by TA cloning.pGEM-T-CDB3 was identified by PCR,restriction enzyme cleavage and gene sequencing.The plasmids p GEM-T-CDB3 and pGEX-4T-1 were digested.The target gene CDB3 and pGEX-4T-1 were recovered,ligated and transformed into E.coli BL21 (DE3) to construct the recombinant plasmid.The expression of target protein was induced by IPTG,while the molecular weight and specificity of the protein were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Results The PCR product of Clostridium difficile toxins B receptor-binding domain (CDB3) was about 1851 bp.Plasmid pGEMT-CDB3 was about 4800 bp,obtained by recombinatiom the cloned vector pGEM-T with CDB3.The recombinant expression plasmid p GEX-4T-1-CDB3 was constructed and the target protein was expressed after induction. Conclusion The prokaryotic expression vector p GEX-4T-1-CDB3 is constructed successfully and the target protein is expressed greatly,which will lay a good foundation for rapid detection of Clostridium difficile toxin B and development of a vaccine.(Chin J Lab Med,2013,36:324-328) Key words: Clostridium difficile; Bacterial proteins; Bacterial toxins; Polymerase chain reaction