• Cloning and expression of PLC-Darpin fusion protein in a prokaryotic host as an immunotoxin putative candidate against breast cancer
  • Arya Sheikhi,1 Mina Shirmohammadpour,2 Samira Shamsi,3 Nima Mahdei Nasirmahalleh,4 Bahman Mirzaei,5,*
    1. Department of Microbiology and Virology, Zanjan University of Medical Sciences
    2. Department of Microbiology and Virology, Zanjan University of Medical Sciences
    3. Department of Microbiology and Virology, Zanjan University of Medical Sciences
    4. Department of Clinical Biochemistry, Zanjan University of Medical Sciences
    5. Department of Microbiology and Virology, Zanjan University of Medical Sciences


  • Introduction: Cancer is one of the genetic diseases, breast cancer (BC) 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 systems by vector, bacterial toxin, and spore. Clostridium novyi (C. novyi), a gram-positive bacillus, is an obligate anaerobe with a spore. This bacterium by producing phospholipase C (PLC) 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 present study aims to clone the PLC-Darpin gene in the pET28a vector and express the protein in the bacteria Escherichia coli (E. coli).
  • Methods: The sequence of the PLC-Darpin gene was amplified by using specific primers with the Polymerase chain reaction (PCR) method, PCR products, and pET28a plasmid was cut with restriction enzymes NdeI and XhoI, and the considered segment was conducted to the cut vector and then transformed into E. coli BL21(DE3) and screened by the antibiotic kanamycin. Colonies were evaluated by PCR method and positive colony plasmid was screened by double digestion method Protein expression was analyzed by using Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and it was checked with specific antibodies by using the 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 the inserted fragment confirmed the enzymatic activity. The cloned fragment was confirmed by using the PCR colony method and the addition of Isopropyl β-d-1-thiogalactopyranoside (IPTG) caused the expression of PLC-Darpin protein, whose size was about 60 kDa, about 3 hours after induction, eventually protein expression by using the western blotting method was confirmed.
  • Conclusion: Due to the enzymatic role and characteristic cytotoxic of PLC in C. novyi, it can create one of the main components of immunotoxin or vaccine.
  • Keywords: PCR, E. coli BL21(DE3), Cloning, Clostridium novyi, SDS-PAGE