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Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella Abortus Rb51strain Publisher



Gheibi A1 ; Khanahmad H6 ; Kardar GA1, 2 ; Boshtam M7 ; Rezaie S3 ; Kazemi B4 ; Khorramizadeh MR5
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Authors Affiliations
  1. 1. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
  2. 2. Department of Immunology, Asthma and Allergy Research Institute, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran
  3. 3. Department of Mycology and Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
  4. 4. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
  5. 5. Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
  6. 6. Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
  7. 7. Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran

Source: Advanced Biomedical Research Published:2019


Abstract

Background: The development of protective vaccines for Brucella spp. has been hampered by the difficulty in transformation of Brucella cells with foreign DNA for genetic manipulation. It seems that the formation of Brucella spheroplasts would increase the efficiency of transformation. The aim of this study was to devise an efficient method for the transformation of Brucella spp. Materials and Methods: At first, spheroplast of Brucella was prepared by glycine and ampicillin induction and transformed using optimized protocols of CaCl2, electroporation, and lipofection methods. Then, the efficacy of transformation was compared between the three-mentioned methods. Results: Ampicillin-induced spheroplasts from early-log phase culture of brucella when incubated in a medium-containing 0.2 M sucrose during cell recovery had higher transformation efficiency in three different methods. Comparison of the transformation efficiency of Brucella abortus RB51 using the CaCl2, lipofection, and electroporation methods revealed that the transformation efficiency with the lipofection method was significantly higher than with other two methods (P < 0.05). Conclusions: Lipofection method by lipofectamine 2000 on ampicillin-induced spheroplasts can be a suitable approach for Brucella transformation. © 2019 Advanced Biomedical Research.
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