Design of vaccine DNA containing genes encoding sicl Escherichia coli antigens Enterotoxigenic (ETEC) and Escherichia coli enterohemorrhagic (EHEC)

Document Type : Original Research

Authors
F. Jafari Parsa 1
S. Zare 2
S. A. Mirhosseini 3
J. Amani 4
1 Department of Genetics, Faculty of Basic Sciences, Islamic Azad University, Research Sciences Branch, Tehran, Iran.
2 Department of Genetics and Biotechnology, Pishva Faculty of Biological Sciences, Islamic Azad University, Varamin Branch, Iran.
3 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran- Iran.
4 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Vanak Sq. Molasadra St. Tehran- Iran.
Abstract
Introduction: Enterotoxigenic Escherichia coli (ETEC) and Enterohemorrhagic Escherichia coli O157:H7 , the most common strains, are causes diarrhea that can kill hundreds of thousands of children annually. The development of an effective combination vaccine for enterohemorrhagic Escherichia coli and Enterotoxigenic Escherichia coli is very important.

Materials and Methods: In this study, the sicl gene was amplified and Subcloned as a DNA vaccine. The sequence of antigen encoding genes was evaluated from the genebank and their epitopes were evaluated to design of primer for the synthetic chimeric gene and was amplified by PCR. Subcloning of a multipartal chimeric gene in eukaryotic expression vector was performed to make a DNA vaccine and finally the protein was purified by nickel chromatography and evaluated by Western blotting.

Results: The immunoblotting results of the expression of SICL chimeric protein indicated the presence of a 76 kDa band in the form of insoluble particles after 12 hours induction. Purification of the recombinant protein using His tag sequencee and confirmation of the purified protein with the recombinant protein specific antibody demonstrated the accuracy of the protein expression.

Discussion & Conclusion: Protein expression, purification and verify by western blotting showed that this recombinant chimeric protein (SICL) can be expressed in eukaryotic host.

Keywords

Subjects

1. Sears, C.L. and J.B. Kaper, Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiological reviews, 1996. 60(1): p. 167.
2. Clarke, S.C., Diarrhoeagenic Escherichia coli—an emerging problem? Diagnostic microbiology and infectious disease, 2001. 41(3): p. 93-98.
3. Brooks, G.F., J.S. Butel, and S.A. Morse, Jawetz, Melnick, & Adelberg's medical microbiology2004: Lange Medical Books/McGraw-Hill, Medical Pub. Division.
4. Karaman, S., J. Cunnick, and K. Wang, Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler’s diarrhea. Plant cell reports, 2012. 31(3): p. 527-537.
5. Bourgeois, A.L., T.F. Wierzba, and R.I. Walker, Status of vaccine research and development for enterotoxigenic Escherichia coli. Vaccine, 2016. 34(26): p. 2880-2886.
6. Schüller, S., Shiga toxin interaction with human intestinal epithelium. Toxins, 2011. 3(6): p. 626-639.
7. Mohawk, K.L., et al., Neutralizing antibodies to Shiga toxin type 2 (Stx2) reduce colonization of mice by Stx2-expressing Escherichia coli O157: H7. Vaccine, 2010. 28(30): p. 4777-4785.
8. Al Jarousha, A.M.K., M.A. El Jarou, and I.A. El Qouqa, Bacterial enteropathogens and risk factors associated with childhood diarrhea. The Indian Journal of Pediatrics, 2011. 78(2): p. 165-170.
9. Navidinia, M., et al., Study prevalence of verotoxigenic E. coli isolated from urinary tract infections (UTIs) in an Iranian children hospital. The open microbiology journal, 2012. 6: p. 1.
10. Moshtagian, F., M. Alipour, and Y. Yahyapour, Prevalence of Escherichia coli pathotypes among children with diarrhea in Babol, Northern Iran. Int J Enteric Pathog, 2016. 4(3): p. 1-4.
11. Rahimi, E., H.R. Kazemeini, and M. Salajegheh. Escherichia coli O157: H7/NM prevalence in raw beef, camel, sheep, goat, and water buffalo meat in Fars and Khuzestan provinces, Iran. in Veterinary Research Forum. 2012. Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
12. DeVinney, R., D.G. Knoechel, and B.B. Finlay, Enteropathogenic Escherichia coli: cellular harassment. Current opinion in microbiology, 1999. 2(1): p. 83-88.
13. Orden, J., et al., Typing of the eae and espB genes of attaching and effacing Escherichia coli isolates from ruminants. Veterinary microbiology, 2003. 96(2): p. 203-215.
14. Torres, A.G., X. Zhou, and J.B. Kaper, Adherence of diarrheagenic Escherichia coli strains to epithelial cells. Infection and immunity, 2005. 73(1): p. 18-29.
15. Khalouie, F., et al., Immunogenic evaluation of chimeric recombinant protein against ETEC, EHEC and Shigella. Molecular Biology Research Communications, 2017. 6(3): p. 101.
16. Jeshvaghani, F.S., et al., Oral immunization with a plant-derived chimeric protein in mice: Toward the development of a multipotent edible vaccine against E. coli O157: H7 and ETEC. Immunobiology, 2019. 224(2): p. 262-269.
17. Karimi Rahjerdi, A., et al., Immunogenic Evaluation of Bivalent Vaccine Candidate against Enterohemorrhagic and Enterotoxigenic Escherichia coli. Iranian Journal of Immunology, 2019. 16(3): p. 200-211.
18. McNeilly, T.N., et al., Escherichia coli O157: H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin. Infection and immunity, 2008. 76(6): p. 2594-2602.
19. Svennerholm, A.-M. and J. Tobias, Vaccines against enterotoxigenic Escherichia coli. Expert review of vaccines, 2008. 7(6): p. 795-804.
20. Odumosu, O., et al., AB toxins: a paradigm switch from deadly to desirable. Toxins, 2010. 2(7): p. 1612-1645.
21. Aslani, M.M. and S. Bouzari, An epidemiological study on Verotoxin-producing Escherichia coli (VTEC) infection among population of northern region of Iran (Mazandaran and Golestan provinces). European journal of epidemiology, 2003. 18(4): p. 345-349.
22. Nataro, J.P. and J.B. Kaper, Diarrheagenic escherichia coli. Clinical microbiology reviews, 1998. 11(1): p. 142-201.
23. Ebrahimi, F., et al., Designing a Recombinant Vaccine Containing Three Bacterial Proteins of EHEC, ETEC, and shigella Dysentery Antigens in E. coli and Evaluation of its Humoral Immunity in Mic. Journal of Mazandaran University of Medical Sciences, 2018. 27(157): p. 1-16.
24. Novinrooz, A., et al., In-silico design, expression, and purification of novel chimeric Escherichia coli O157: H7 OmpA fused to LTB protein in Escherichia coli. PLoS One, 2017. 12(3): p. e0173761.
25. Bentancor, L.V., et al., A DNA vaccine encoding the enterohemorragic Escherichia coli Shiga-like toxin 2 A2 and B subunits confers protective immunity to Shiga toxin challenge in the murine model. Clin Vaccine Immunol, 2009. 16(5): p. 712-8.
Pathobiology Reserach
Volume 23, Issue 4
June 2020
Pages 1-8