Cloning and Expression of N-terminal Region of IpaD from Shigella dysenteriae in E. coli

Mojtaba Saadati, Mohammad Heiat, Shahram Nazarian, Babak Barati, Hossein Honari, Mohammad Doroudian, Mahdi Hesaraki, Fatemeh Malaei, Marzieh Eghtedardoust, Mohammad Rahbar, Saeed Hosseinzadeh, Mehrdad Hashemi

Abstract


214

Genus Shigella is one of the important members of the family enterobacteriacae. There are numerous antigens in Shigella carrying by a 220 kb plasmid. Among them, IpaD is the key virulence factor of S. flexneri. Apart from having effectors function that is essential for host cell invasion and intracellular survival, this protein also controls the secretion and translocation of other effector proteins into eukaryotic host cells. In the present study, we have cloned and expressed the ipaD in E. coli. The ipaD gene was amplified by PCR. Prokaryote expression vector pET-28a(+)- ipaD was constructed, and used to transform E. coli BL21DE3 plySs. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of IpaD-His by a rabbit monoclonal antibodies against his-tag. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced IpaD at the 1 mmol/L of IPTG. IpaD protein was able to react with the rabbit monoclonal antibody against His-tag.  IpaD is essential for Shigella spp invasion. N-terminal region is most significant functional fragment of IpaD. Purification of IpaD from the wild type of Shigella is difficult furthermore profound study on a specific domain on the N-terminal of IpaD by using the wild type of purified IpaD is not feasible.

 


Keywords


Shigella ssp; N-terminal region, ipaD; cloning; expression

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DOI: https://doi.org/10.22037/jps.v1i4.2225

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"Journal of Paramdedical Sciences", is a publication of "School of Paramedical Sciences, Shahid Beheshti University of Medical Sciences" and "Iranian Society of Medical Proteomics".

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EISSN: 2008-4978

PISSN: 2008-496X