Volume 22, Issue 1 (4-2019)
J Arak Uni Med Sci 2019, 22(1): 96-107 |
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Roghanian P, Amani J, Zare S, Nour Mohammadi Z. Design, Construction, Expression and Purification of the Gene Encoding Chimeric Cfae, Cotd Protein, from Enterotoxigenic Escherichia Coli. J Arak Uni Med Sci 2019; 22 (1) :96-107
URL: http://jams.arakmu.ac.ir/article-1-5831-en.html
URL: http://jams.arakmu.ac.ir/article-1-5831-en.html
1- Department of Genetics, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2- Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran. , jafar.amani@gmail.com
3- Department of Genetics and Biotechnology, School of Biological Science, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran.
2- Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran. , jafar.amani@gmail.com
3- Department of Genetics and Biotechnology, School of Biological Science, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran.
Abstract: (3049 Views)
Background and Aim: Enterotoxigenic Escherichia coli (ETEC) is one of the most common bacterial causes of diarrhea deaths among children and travelers in developing countries. The ETEC colonization factors, such as CFA/I and CS2 play an important role in the development of the disease. In this study, to produce the CFaE fusion recombinant protein, the tip subunits CFA/I(CfaE) and sub structural unit of CS2 (CotD) from ETEC, were used. Since mucosal immune responses to CFs can prevent disease, the aim of this study was to develop a chimeric antigen for developing the effective vaccine.
Materials and Methods: In order to amplify the cfae-cotd gene, a dual gene construct consisting of cfae and cotd, the PCR reaction was performed by designed primers. The propagated gene was cloned in the expression vector pET28a. Following the induction of a recombinant gene construct with IPTG, the recombinant protein was expressed and purified by Ni-NTA chromatography column and confirmed by western blotting by Anti-Histag.
Ethical Considerations: This study with research ethics code IR.IAU.SRB.REC.1397.066 has been approved by research ethics committee at Islamic Azad University, Science and Research Branch of Tehran, Iran.
Findings: Cloning accuracy was confirmed by PCR and enzyme digestion reaction. The presence of the band in the SDS-PAGE 10% gel in the 68 kDa region, the expression of the recombinant protein, and the presence of the band on the nitrocellulose paper in the Western blotting test confirmed the production of recombinant protein.
Conclusion: Optimization of codon and expression in heterologous hosts is a useful method for the production of recombinant proteins. The production of ETEC antigens as a candidate for vaccination against this bacterium is also prominent.
Materials and Methods: In order to amplify the cfae-cotd gene, a dual gene construct consisting of cfae and cotd, the PCR reaction was performed by designed primers. The propagated gene was cloned in the expression vector pET28a. Following the induction of a recombinant gene construct with IPTG, the recombinant protein was expressed and purified by Ni-NTA chromatography column and confirmed by western blotting by Anti-Histag.
Ethical Considerations: This study with research ethics code IR.IAU.SRB.REC.1397.066 has been approved by research ethics committee at Islamic Azad University, Science and Research Branch of Tehran, Iran.
Findings: Cloning accuracy was confirmed by PCR and enzyme digestion reaction. The presence of the band in the SDS-PAGE 10% gel in the 68 kDa region, the expression of the recombinant protein, and the presence of the band on the nitrocellulose paper in the Western blotting test confirmed the production of recombinant protein.
Conclusion: Optimization of codon and expression in heterologous hosts is a useful method for the production of recombinant proteins. The production of ETEC antigens as a candidate for vaccination against this bacterium is also prominent.
Type of Study: Original Atricle |
Subject:
Basic Sciences
Received: 2018/06/30 | Accepted: 2018/12/19
Received: 2018/06/30 | Accepted: 2018/12/19
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