Majallah-i Dānishgāh-i ̒Ulūm-i Pizishkī-i Qum (Sep 2015)
Cloning and Recombinant Expression of Intimin C-Terminal Domain of EHEC Pathogen in E. coli BL21(DE3) Strain
Abstract
Abstract Background and Objectives: Some intestinal infections are caused by pathogens that enter the gastrointestinal tract of humans by consumption of contaminated food. A set of proteins are involved in colonization of E. coli O157:H7 in the large intestine, some of which initiate this phenomenon and their absence prevents it. The aim of this study was cloning and recombinant expression of C-terminal polypeptide of intimin, with a length of 300 amino acids, as a vaccine candidate against E. coli O157:H7. Methods: Specific primers for eae gene were designed using Oligo software version 7.0 and gene amplification was performed by PCR from template DNA. Enzymatic digestion and gene ligation were performed in pET28a(+) vector. Recombinant expression of intimin was done by IPTG inducer following the cloning confirmation. Western blotting analysis was performed to confirm the intimin protein. Results: Cloning of eae gene in the pET28a (+) vector was performed appropriately among desired sites, and intimin protein had a proper and significant recombinant expression after the induction. Also, the antibody used in western blotting could identify intimin. Conclusion: In this study, a stable construct containing eae gene was synthetized, which showed an appropriate recombinant expression of intimin protein. Therefore, this construct can be used to produce intimin protein for immunization studies against E. coli O157:H7.
