The cloning of Clostridium difficile toxin A gene

In the past twenty years, there has been a significant increase in incidence and mortality of both hospital acquired and community acquired Clostridium difficile. Toxin A (enterotoxin) and toxin B (cytotoxin) are responsible for the pathogenesis of the disease. Moreover, binary toxin, the cause of hospital outbreaks, has recently been identified. The current study aimed to produce the antigen that can be used in diagnostic tests by cloning C. difficile toxin A gene. In our study, tcdA positive strain, C. difficile 06012 obtained from Turkey Public Health Association (Ankara) was used. Specific primers were designed according to the reference sequence in Genbank NC-009089.1 and modified by adding Bam HI restriction site to the end. DNA was isolated from C. difficile strain and by using Extaq polymerase enzyme, DNA fragment including tcdA gene was amplified. Amplicon and pUC19 plasmid were digested with BamHI and ligated, than it was used to transform E. coli DH10B strain by electroporation. Recombinant plasmids were studied and the plasmid that contained tcdA gene was chosen and stored for further studies. In this study, C. difficile tcdA gene was cloned for the synthesis of recombinant toxin. The synthesis and purification of toxin A by recombinant technology will be helpful for further studies. Using purified toxin production of polyclonal and monoclonal antibodies and, determining serum antibody levels, conducting studies on in vitro and in vivo enzyme activity will be possible.