Cloning and Recombinant Expression of EspA as a Virulence Factor of E. coli O157:H7

Bakhshi, Mostafa and Ebrahimi, Firouz and Zargan, Jamil and Nazarian, Shahram and Sheikhzade, Vahideh (2014) Cloning and Recombinant Expression of EspA as a Virulence Factor of E. coli O157:H7. Journal of Mazandaran University of Medical Sciences, 24 (117). pp. 12-20.

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Abstract Background and purpose: E. coli O157:H7 is one of the intestinal pathogens that mediate serious damages in gastrointestinal track. The bacterium attachment in large intestine is mediated via some colonization factors mainly Type III secretion proteins that are involved in this process. Among these factors, EspA protein plays an important role in system foundation. The aim of this study was cloning and recombinant expression of EspA in order to achieve a stable construct for the protein production and using it as a vaccine candidate in future investigations. Materials and methods: Specific primers were designed by Oligo 7 software and gene amplification was performed by PCR technique on template DNA. Enzymatic digestion and ligation were done by restriction enzymes and T4 ligase enzyme, respectively. Recombinant expression of EspA was induced by IPTG following cloning confirmation. Protein confirmation was done by Western blotting analyze. Results: Results showed that cloning of espA in pET28a (+) vector was performed in an appropriate mode between expected sites. Also, EspA had good and remarkable recombinant expression after induction and the antibody used in western blotting could recognize EspA on nitrocellulose membrane. Conclusion: Stable recombinant construct containing espA gene was fabricated that showed appropriate recombinant expression of protein. So, this protein could be used in future studies as a vaccine candidate against E. coli O157:H7.

Item Type: Article
Uncontrolled Keywords: Intestinal pathogen, espA gene cloning, recombinant expression, Western blotting
Depositing User: Unnamed user with email
Date Deposited: 04 Jan 2018 18:56
Last Modified: 04 Jan 2018 18:56

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