Cloning and expression of toxin C gene of Clostridium difficile and its polyclonal antibody preparation / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To clone and express toxin C (tcdC) gene pathogenicity locus of Clostridium difficile (CD), and prepare the polyclonal antibody against TcdC protein. METHODS The tcdC gene was amplified from CD (ATCC43255) strain genome DNA and inserted into prokaryotic expression vectors. The correct recombinant plasmids were transformed into E.coli which was induced to express the GST-TcdC/L and IL1-TcdC/L fusion protein. Meanwhile, the fusion proteins were respectively purified through Ni-NTA agarose affinity chromatography and Q-Sepharose Fast Flow. The purified GST-TcdC/L fusion protein was used as an antigen to inoculate rabbits to produce antiserum. Two weeks after the final immunization, the rabbits were sacrificed and serum was collected. The titer of the serum was deter-mined by ELISA and the reactivity of the polyclonal antibody was identified by Western blotting. RESULTS The SDS-PAGE result showed that GST-TcdC/L and IL1-TcdC/L fusion proteins were expressed at the size of 45 ku and 39 ku. The titer of TcdC/L polyclonal antibody was over 16.4 × 104. Western blotting detection demonstrated that the TcdC/L polyclonal antibody recognized the TcdC protein in CD (ATCC43255). CONCLUSION The tcdC/L gene is cloned and the polyclonal antibody against TcdC/L is prepared, which will contribute to studing the function and mechanism of tcdC gene in pathogenicity locus.