Molecular cloning and biologically active production of IpaD N-terminal region |
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Authors: | Mahdi Hesaraki Mojtaba Saadati Hossein Honari Gholamreza Olad Mohammad Heiat Fatemeh Malaei Reza Ranjbar |
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Institution: | 1. Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran;2. Department of Biology, Faculty of Basic Sciences, Imam Hossein University (IHU), Tehran, Iran;3. Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran |
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Abstract: | Shigella is known as pathogenic intestinal bacteria in high dispersion and pathogenic bacteria due to invasive plasmid antigen (Ipa). So far, a number of Ipa proteins have been studied to introduce a new candidate vaccine. Here, for the first time, we examined whether the N-terminal region of IpaD72–162 could be a proper candidate for Shigella vaccine. Initially, the DNA sequence coding N-terminal region was isolated by PCR from Shigella dysenteriae type I and cloned into pET-28a expression vector. Then, the heterologous protein was expressed, optimized and purified by affinity Ni–NTA column. Western blot analysis using, His-tag and IpaD72–162 polyclonal antibodies, confirmed the purity and specificity of the recombinant protein, respectively. Subsequently, the high immunogenicity of the antigen was shown by ELISA. The results of the sereny test in Guinea pigs showed that IpaD72–162 provides a protective system against Shigella flexneri 5a and S. dysenteriae type I. |
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Keywords: | IpaD Sereny test Protectivity |
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