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:: Volume 8, Issue 4 (Winter 2019) ::
J Fasa Univ Med Sci 2019, 8(4): 1025-1035 Back to browse issues page
Cloning and evaluation of gene expression and purification of gene encoding recombinant protein containing binding subunit of coli surface antigens CS1 and CS2 from Enterotoxigenic Escherichia coli
Dorna Khoobbakht1 , Jafar Amani 2, Shohreh Zare3 , Zahra Nourmohammadi1
1- Department of Genetics, Faculty of Basic Sciences, Islamic Azad University, Research Branch, 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
Abstract:   (1295 Views)
Background & Objective: Enterotoxigenic Escherichia coli (ETEC) is a major causative agent of diarrhea. Enterotoxins and the colonization factors (CFs) are major virulence factors in ETEC infections. The bacterium binds to the intestinal epithelial cell surface through colonization factors and produces enterotoxins that cause excessive fluid and electrolyte secretion in the lumen of the intestine, which ultimately leads to diarrhea. Due to the difficult of treatment and the high prevalence of this disease, the design of the vaccine against this organism is one of the goals of the World Health Organization. The CooD-CotD protein, as adhesion tip subunits of CS1 and CS2, plays an important role in bacterial attachment to the intestinal epithelial cells. In this study, the expression and purification of chimeric protein CooD-CotD was carried out with the aim of investigating as candidate vaccine.
Materials & Methods: In this study, codon optimization of cooD-cotD chimeric gene was performed by Gene Designer software. The gene was amplified by PCR and cloned into pJET1.2 / blunt then it was subcloned into pET28a to express chimeric protein. Recombinant protein was purified following expression, using Ni-NTA affinity chromatography and confirmed by western blotting analysis.
Results: The presence of 82kDa in the SDS-PAGE gel showed that expression of CooD-CotD chimeric protein and confirmed by western blotting analysis. Yield of purified protein was 121 mg/ml.
Conclusions: Expression and protein purification studies showed that this protein has expression in the homologous host.
Keywords: Enterotoxigenic Escherichia coli, CS1, CS2, Recombinant expression
Full-Text [PDF 1202 kb]   (175 Downloads)    
Type of Study: Research | Subject: Medical Biotechnology
Received: 2018/06/27 | Accepted: 2018/12/10 | Published: 2019/03/17
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Khoobbakht D, Amani J, Zare S, Nourmohammadi Z. Cloning and evaluation of gene expression and purification of gene encoding recombinant protein containing binding subunit of coli surface antigens CS1 and CS2 from Enterotoxigenic Escherichia coli. J Fasa Univ Med Sci. 2019; 8 (4) :1025-1035
URL: http://journal.fums.ac.ir/article-1-1770-en.html


Volume 8, Issue 4 (Winter 2019) Back to browse issues page
Journal of Fasa University of Medical Sciences
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