Journal of Advances in Medical and Biomedical Research

en Design, Cloning, and Expression of PLC-Darpin Fusion Protein as Putative Immunotoxin in a Prokaryotic Host Life Science Life Science مقاله پژوهشی Original Research Article Background & Objective:  Cancers are common genetic disease that can cause death. Bacteria, such as Clostridium novyi, potentially could be used in cancer treatment by producing an enzyme called phospholipase C (PLC), which causes cell lysis. The aims of this study are to cloning and expression of PLC- Darpin in prokaryotic host.  Materials & Methods:  Briefly, the PLC- Darpin gene sequence was amplified using PCR. The amplified fragment was conducted into the pET28a vector, transformed into E. coli BL21 (DE3), and screened by the double digestion method. Protein expression was analyzed using SDS-PAGE and checked with specific antibodies using the western blotting method. The cloned fragment was confirmed using the PCR colony method. Results:  Designed PLC- Darpin protein sequence (1600 bp) was successfully amplified by specific primers taking advantage of PCR method. Then, cloned PLC- Darpin candidate sequence was reconfirmed by digestion procedure, and colony PCR. Finally, 60 KDa expressed protein in prokaryotic host reconfirmed by SDS- page and western blotting. Conclusion:  Fused PLC enzyme to Darpin protein could be considered as a main putative immunotoxin due to its enzymatic role and cytotoxicity which may be utilized as a therapeutic choice in the future with further investigations.   Immunotoxin, Gene cloning, Darpin, Fusion protein, PLC-Darpin 477 488 http://journal.zums.ac.ir/browse.php?a_code=A-10-5288-7&slc_lang=en&sid=1 Samira Shamsi samira shams 5200319475328460082359 5200319475328460082359 No Department of Microbiology and Virology, Zanjan University of Medical Sciences, Zanjan, Iran Nima Mahdei Nasirmahalleh nima.mahdaii@gmail.com 5200319475328460082360 5200319475328460082360 No Student Research Committee, Department of Medical Microbiology and Virology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran 3Student Research Committee, Department of Medical Biochemistry, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran mina Shirmohammadpour mmina.shmp@gmail.com 5200319475328460082361 5200319475328460082361 No Student Research Committee, Department of Medical Microbiology and Virology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran 3Student Research Committee, Department of Medical Biochemistry, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran Davoud Afshar dr.davodafshar@gmail.com 5200319475328460082362 5200319475328460082362 No Department of Microbiology and Virology, Zanjan University of Medical Sciences, Zanjan, Iran Bahman Mirzaei dr.bahman.m@gmail.com 5200319475328460082363 5200319475328460082363 Yes Department of Microbiology and Virology, Zanjan University of Medical Sciences, Zanjan, Iran