Cloning and Expression of Twin-Arginine Translocation D Family Deoxyribonuclease of Clostridium Chauvoei

摘要

Clostridium chauvoei, an anaerobic bacterium reported worldwide, is responsible for Black Quarter, a dreadful disease of ruminants. This bacterium produces many toxins responsible for the pathogenesis of the disease. Except for the well-studied virulence factors such as cctA, flagellin andsialidase genes, the exact role of other toxins of C. chauvoei remains unknown. This necessitates studies on the activities of the C. chauvoei toxins and virulence. In the present study, Twin-Arginine Translocation D (TatD) family deoxyribonuclease of the bacterium was selected. The tatD gene C. chauvoei was amplified by PCR and cloned into p-Rham-N-His-SUMO-Kan expression vector, followed by transformation into the E.cloni 10G competent cells. Clones obtained were confirmed by colony PCR. These tatDclones were sequenced and analysed phylogenetically, which revealed the close relationship of C. chauvoei strain to C. isatidis, C. saccharobutylicum, C. botulinum and C. taeniosporum based on tatD sequence analysis. Upon induction of the clones with L-rhamnose, the protein expression was obtained at 42.3 kDa and the same was further confirmed by Western blotting.