Design and recombinant expression of STX1B-IpaD immunogenic protein from Enterohemorrhagic Escherichia coli and Shigella

Mosadegh, Shadi; Abtahi, Hamid; Amani, Jafar; Zare Karizi, Shohreh; Salmanian, A.H.

Design and recombinant expression of STX1B-IpaD immunogenic protein from Enterohemorrhagic Escherichia coli and Shigella

Document Type : Original Research

Authors

Shadi Mosadegh ¹

Hamid Abtahi ²

Jafar Amani ³

Shohreh Zare Karizi ⁴

A.H. Salmanian ⁵

¹ Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

² Molecular and Medicine Research Center, Arak University of Medical Sciences, Arak, Iran

³ Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences

⁴ Department of Biology, Varamin Pishva Branch, Islamic Azad University, Pishva, Varamin, Iran

⁵ Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB)

Abstract

Background: Shigella and Enterohemorrhagic Escherichia coli are among the most common causes of bacterial diarrhea, and no effective vaccine candidate for these bacteria have approved yet. Due to the role of IpaD protein and Shigella enterotoxin B subunit (StxB) in Shigella and E. coli O157: H7 pathogenicity, STX1B-IpaD chimeric protein can be used as a suitable molecule to produce a recombinant vaccine candidate. This study aimed to clone, express, and purify STX1B-IpaD chimeric protein to develop an effective vaccine candidate against Shigella and E. coli O157: H7 species. Materials and Methods: IpaD gene with NdeI and BamHI restriction enzyme sites was isolated from a recombinant vector and subcloned into the pET28a -STX1B expression vector. Vector was transferred to E.coli strain Rosetta (DE3) and confirmed by PCR and restriction enzyme digestion. SDS-PAGE and western blotting were used to confirm the recombinant protein. The recombinant STX1B-IpaD protein was purified by affinity chromatography, and its concentration was measured by the Bradford method. Results: The PCR and restriction enzyme digestion showed the accuracy of the gene cloning. The protein electrophoresis showed the proper expression and correct molecular weight (27 kDa) of STX1B-IpaD. The western blot analysis confirmed the recombinant protein. The recombinant protein concentration was estimated at more than 0.3 gr/L. Conclusion: An effective method for the production of recombinant proteins is codon optimization and effective expression in heterologous hosts. After the immunogenicity in the animal model, this recombinant protein can be used as a chimeric vaccine candidate against EHEC and Shigella bacteria.

Keywords

Shigella

E.coli O157:H7

invasion plasmid antigen D (IpaD)

Shigella enterotoxin B subunit (StxB)

20.1001.1.23222115.1401.13.4.10.9

Subjects

Microbial biotechnology

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