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Cloning virG gene and generation mutant construct of pGEM∆virG in order to induction recombination in native Shigella dysenteriae
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Hora Ahmadi Danesh1   , Mojtaba Saadati2 , Mohammad Doroudian3 , Bagher Yakhchali4 , Ali Asghar Karkhaneh5 , Sayed Mostafa Hosseini6 , Mokhdar Zare7 , Mehrdad Hashemi8 |
1- MSc Student of Cellular and Molecular Biology, Department of Cell Biology, Faculty of Science, Imam Hossein University, Tehran, Iran
2- PhD of Bacteriology, Department of Cell Biology, Faculty of Science, Imam Hossein University, Tehran, Iran , m_saadati@yahoo.com
3- MSc Student of Cellular and Molecular Biology, Islamic Azad University, Central Tehran Branch, Young Reserchers Club, Tehran, Iran
4- PhD of Microbiology, Department of Industrial and Environmental Biotechnology, National Institute of Genetics and Biotechnology, Tehran, Iran
5- PhD of Molecular Genetics, Department of Industrial and Environmental Biotechnology, National Institute of Genetics and Biotechnology, Tehran, Iran
6- Young Researchers Club, Islamic Azad University, Science and Research Campus, Tehran, Iran
7- Researcher, Department of Cell Biology, Faculty of Science, Imam Hossein University, Tehran, Iran
8- PhD of Molecular Genetics, Islamic Azad University, Tehran Medical Branch, Tehran, Iran |
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Abstract: (17593 Views) |
Background: Shigellosis disease is the major causes of morbidity in children with diarrhea in Iran. The virG (icsA) gene plays a key role in pathogenesis and ability of invasion in shigella. The aim of this study was cloning, sequencing virG gene and developing a mutant construct pGEM∆virG in order to induction recombination in a native shigella for generation a live attenuated vaccine candidate strain.
Materials and methods: Initially, by use of biochemical tests, the native shigella strain was detected. The virG gene was cloned in pGEM-7zf vector and the nucleotide sequence was determined. According to the data of sequencing, digestion mapping of pGEMvirG vactor was obtained and a part of virG gene by using enzymatic digestion was removed. Finally, pGEM∆virG construct was transformed to E. coli by utilization of chemical transformation method.
Results: The native shigella strain by using biochemical tests was confirmed. The result of sequencing virG gene (native strain) was submitted in NCBI Genebank database. The pGEM∆virG construct contains a mutant construct of virG gene which 1751 bp was deleted through enzymatic digestion reaction and transformed in E. coli.
Conclusion: Using the technique of allelic exchange based on the incident of recombination in bacteria is one of the most effective methods to develop a disruption in the target genes. This mutant construct can be applied in development of a live attenuated Shigella dysenteriae vaccine candidate. |
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| Keywords: Allelic exchange, Shigella dysenteriae, virG gene, Live attenuated vaccine candidate |
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Full-Text [PDF 401 kb]
(3330 Downloads)
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Semi-pilot: Experimental |
Subject:
Molecular Biology
Received: 2012/09/23 | Published: 2012/09/15
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