• Cloning and expression of Clostridium novyi phospholypase C toxin in prokaryotic host
  • Mina Shirmohammadpour,1 Samira Shamsi,2 Nima Mahdaei Nasirmahalleh,3 Bahman Mirzaei,4,*
    1. Department of Microbiology and Virology, Zanjan University of Medical Sciences, Zanjan, Iran
    2. Department of Microbiology and Virology, Zanjan University of Medical Sciences, Zanjan, Iran
    3. Department of Clinical Biochemistry, Zanjan University of Medical Sciences, Zanjan, Iran
    4. Department of Microbiology and Virology, Zanjan University of Medical Sciences, Zanjan, Iran


  • Introduction: Cancer is one of the genetic diseases, breast cancer is one of the commonest of them and the first cause of death in women. Bacteria are used in different ways in cancer treatment such as: gene transfer system by vector, bacterial toxin and spore. Clostridium novyi, a gram-positive bacillus, is an obligate anaerobes with spore. This bacterium by producing phospholipase C enzyme and interacting with the membrane of eukaryotic cells causes cell lysis. Due to the cytotoxic characteristic and role of the enzyme in the pathogenesis of the disease, It can establish one of the main components of immunotoxin or vaccine. The aim of the present study is cloning the PLC-Darpin gene in the pET28a vector and express the protein in the bacteria of Escherichia coli.
  • Methods: The sequence of PLC-Darpin gene was amplified by using specific primers with PCR method, PCR products and pET28a plasmid were cut with restriction enzymes NdeI and XhoI and the considered segment was conjuncted to the cut vector and then transformed into Ecoli BL21(DE3) and screened by the antibiotic of canamaycin. Colonies were evaluated by PCR method and positive colony plasmid was screened by double digestion method and Protein expression was analyzed by using SDS-PAGE gel, Finally it was checked with specific antibody by using western blotting method.
  • Results: The size of the amplified fragment which is about 1600 bp was confirmed by PCR and using agarose gel. The cut plasmid pET28a after inserted fragment confirmed the enzymatic activity. The cloned fragment was confirmed by using the PCR colony method and the addition of IPTG caused the expression of PLC-Darpin protein, which size was about 60 kDa, about 3 hours after induction, eventually protein expression by using western blotting method was confirmed.
  • Conclusion: Due to the enzymatic role and characteristic cytotoxic of phospholipase C in Clostridium novi, it can create one of the main components of immunotoxin or vaccine.
  • Keywords: E. coli BL21(DE3), Cloning, Clostridium Novyi, SDS-PAGE, Western blotting