Expression of Recombinant Chimeric EspA-intimin Protein in Nicotiana tobaccum for Oral Vaccine Development

Sahshorpour, Mahdieh; Amani, Jafar; Jafari, Mahyat; Salmanian, Ali Hatef

doi:10.29252/pgr.1.1.77

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Volume 1, Issue 1 (2014)

pgr 2014, 1(1): 77-94

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Expression of Recombinant Chimeric EspA-intimin Protein in Nicotiana tobaccum for Oral Vaccine Development

Mahdieh Sahshorpour

, Jafar Amani

, Mahyat Jafari

, Ali Hatef Salmanian ^*

Associate Professor, Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran

Abstract: (26415 Views)

One of the important pathogens which cause hemorrhagic colitis and hemolytic uremic syndrome in humans is enterohemorrhagic Escherichia coli (EHEC) O157:H7. Cattle are the most important reservoir of this bacterium. EspA and Intimin are two protein factors for bacteria colonization on intestinal epithelium and cause attaching/effacing lesion. The LEE pathogenicity islands code these proteins. EspA is part of type III secretion systems which delivers Tir to the host cell and integrate to membrane. Intimin encoded by eae gene and fused to Tir. In this research we supposed that chimeric recombinant form of EI gene containing EspA and Intimin were fused with a linker as an edible candidate vaccine would reduce colonization of E. coli O157:H7 in animal model. We constructed a synthetic gene EspA (E120) and intimin (Int282) fused by (EAAAK)4 sequence. The synthetic gene (EI) was codon optimized and subcloned into plant expression vector (PBI121) under CaMV35S promoter and then transferred to tobacco plant by agrobacterium mediated protocol. The presence of inserted gene in plant genome was documented by PCR and RT-PCR methods. The amount of EI protein in transgenic tobacco leaves were estimated 0.1% of the total soluble protein (TSP) by ELISA method.

Keywords: E. coli O157:H7, Edible vaccine, EspA, Intimin, Transgenic plant

Full-Text [PDF 977 kb] (4893 Downloads)

Type of Study: Research | Subject: Plant improvement

References

1. Adu-Bobie, J., Frankel, G., Bain, C., Goncalves, A.G., Trabulsi, L.R., Douce, G., Knutton, S. and Dougan, G. (1998). Detection of intimins α, β, γ, and δ, four intimin derivatives expressed by attaching and effacing microbial pathogens. Journal of Clinical Microbiology, 36: 662-668.

2. Allen, K.J., Rogan, D., Finlay, B.B., Potter, A.A. and Asper, D.J. (2011). Vaccination with type III secreted proteins leads to decreased shedding in calves after experimental infection with Escherichia coli O157. Canadian Journal of Veterinary Research, 75: 98-105.

3. Amani, J., Mousavi, S.L., Rafati, S. and Salmanian, A.H. (2009). In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications. Theoretical Biology and Medical Modelling, 6: 28-38.

4. Amani, J., Mousavi, S.L., Rafati, S. and Salmanian, A.H. (2011). Immunogenicity of a plantderived edible chimeric EspA, Intimin and Tir of Escherichia coli O157: H7 in mice. Plant Science,180: 620-627.

5. Amani, J., Salmanian, A.H., Rafati, S. and Mousavi, S.L. (2010). Immunogenic properties of chimeric protein from espA,eae and tirgenes of Escherichia coli O157: H7. Vaccine, 28: 6923-6929.

6. Arai, R., Ueda, H., Kitayama, A., Kamiya, N. and Nagamune, T. (2001). Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Engineer Design and Selection, 14: 529-532.

7. Aslani, M.M. and Bouzari, S. (2003). 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, 18: 345-349.

8. Carvalho, H.M., Teel, L.D., Kokai-Kun, J.F. and O'Brien, A.D. (2005). Antibody against the carboxyl terminus of intimin α reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization. Infection and Immunity, 73: 2541-2546.

9. Cook, S., Maiti, P., DeVinney, R., Allen‐Vercoe, E., Bach, S. and McAllister, T. (2007). Avian and mammalian derived antibodies against adherence associated proteins inhibit host cell colonization by Escherichia coli O157: H7. Journal of Applied Microbiology, 103: 1206-1219.

10. Dean-Nystrom, E.A., Bosworth, B.T., Moon, H.W. and O’brien, A.D. (1998). Escherichia coli O157: H7 requires intimin for enteropathogenicity in calves. Infection and Immunity, 66: 4560-4563.

11. Dziva, F., Van Diemen, P.M., Stevens, M.P., Smith, A.J. and Wallis, T.S. (2004). Identification of Escherichia coli O157: H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis. Microbiology, 150: 3631-3645.

12. Fan, H.Y., Wang, L., Luo, J. and Long, B.G. (2012). Protection against Escherichia coli O157: H7 challenge by immunization of mice with purified Tir proteins. Molecular Biology Reports, 39: 989-997.

13. Garmendia, J., Frankel, G. and Crepin, V.F. (2005). Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. Infection and Immunity, 73:2585-2573.

14. Jerse, A. and Kaper, J. (1991). The eae gene of enteropathogenic Escherichia coli encodes a 94- kilodalton membrane protein, the expression of which is influenced by the EAF plasmid. Infection and Immunity, 59: 4302-4309.

15. Judge, N.A., Mason, H.S. and O'Brien, A.D. (2004). Plant cell-based intimin vaccine given orally to mice primed with intimin reduces time of Escherichia coli O157: H7 shedding in feces. Infection and Immunity, 72: 168-175.

16. Kahrizi, D., Salmanian, A.H., Afshari, A., Moieni, A. and Mousavi, A. (2007). Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E. coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate. Plant Cell Reports, 26: 95-104.

17. Kang, T.J., Han, S.C., Jang, M.O., Kang, K.H., Jang, Y.S. and Yang, M.S. (2004). Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence. Applied Biochemistry and Biotechnology, 117: 175-187.

18. Karimi, F., Mousavi, A., Salmanian, A.H., Alizadeh, H. and Rafati, S. (2013). Immunogenicity of EIT chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against Escherichia coli O157: H7. Plant Biotechnology Reports, 7: 535-546.

19. Karmali, M.A. (1989). Infection by verocytotoxin-producing Escherichia coli. Clinical Microbiology Reviews, 2: 15-38.

20. Khaitsa, M.L., Smith, D.R., Stoner, J.A., Parkhurst, A.M., Hinkley, S., Klopfenstein, T.J. and Moxley, R.A. (2003). Incidence, duration, and prevalence of Escherichia coli O157:H7 fecal shedding by feedlot cattle during the finishing period. Journal of Food Protection, 66: 1972-2179.

21. La Ragione, R.M., Patel, S., Maddison, B., Woodward, M.J., Best, A., Whitelam, G.C. and Gough, K.C. (2006). Recombinant anti-EspA antibodies block Escherichia coli O157: H7-induced attaching and effacing lesions in vitro. Microbes and Infection, 8: 426-433.

22. Lal, P., Ramachandran, V., Goyal, R. and Sharma, R. (2007). Edible vaccines: current status and future. Indian Journal of Medical Microbiology, 25: 93-102.

23. McNeilly, T.N., Mitchell, M.C., Nisbet, A.J., McAteer, S., Erridge, C., Inglis, N.F., Smith, D.G., Low, J.C., Gally, D.L. and Huntley, J.F. (2010a). IgA and IgG antibody responses following systemic immunization of cattle with native H7 flagellin differ in epitope recognition and capacity to neutralise TLR5 signalling. Vaccine, 28: 1412-1421.

24. McNeilly, T.N., Mitchell, M.C., Rosser, T., McAteer, S., Low, J.C., Smith, D.G., Huntley, J.F., Mahajan, A. and Gally, D.L. (2010b). Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157: H7 following oral challenge. Vaccine, 28: 1422-1428.

25. Moon, H., Whipp, S., Argenzio, R., Levine, M. and Giannella, R. (1983). Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infection and Immunity, 41: 1340-1351.

26. Moravec, T., Schmidt, M.A., Herman, E.M. and Woodford Thomas, T. (2007). Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine. Vaccine, 25: 1647-1657.

27. Murphy, M., Buckley, J., Whyte, P., O’Mahony, M., Anderson, W., Wall, P. and Fanning, S. (2007). Surveillance of dairy production holdings supplying raw milk to the farmhouse cheese sector for Escherichia coli O157, O26 and O111. Zoonoses and Public Health, 54: 358-365.

28. Oliveira, A.F., Cardoso, S.A., Almeida, F.B., De-Oliveira, L.L., Pitondo-Silva, A., Soares, S.G .and Hanna, E.S. (2012). Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice. Microbiol and Immunology, 56: 513-522.

29. Potter, A.A., Klashinsky, S., Li, Y., Frey, E., Townsend, H., Rogan, D., Erickson, G., Hinkley, S , Klopfenstein, T. and Moxley, R.A. (2004). Decreased shedding of Escherichia coli O157: H7 by cattle following vaccination with type III secreted proteins. Vaccine, 22: 362-369.

30. Roe, A.J., Yull, H., Naylor, S.W., Woodward, M.J., Smith, D.G. and Gally, D.L. (2003). Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157: H7 is controlled at the posttranscriptional level. Infection and Immunity, 71: 5900-5909.

31. Rossi, L., Di Giancamillo, A., Reggi, S., Domeneghini, C., Baldi, A., Sala, V., Dell'Orto, V., Coddens, A., Cox, E. and Fogher, C. (2013). Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model. Journal of Veterinary Science, 14: 263-270.

32. Sedighianrad, H., Mousavi, S.L., Rasooli, I., Amani, J. and Jalali-Nadooshan, M.R. (2013). EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7. Iranian Journal of Microbiology, 5(3): 244-251.

33. Shekarforoush, S., Tahamtan, Y. and Pourbakhsh, A. (2008). Detection and frequency of Stx2 gene in Escherichia coli O157 and O157: H7 strains isolated from sheep carcasses in Shiraz-Iran. Pakistan Journal of Biological Sciences, 11: 1085-1092.

34. Stevens, M.P., Van Diemen, P.M., Dziva, F., Jones, P.W. and Wallis, T.S. (2002). Options for the control of enterohaemorrhagic Escherichia coli in ruminants. Microbiology, 148: 3767-3778.

35. Stewart, C.S. and Flint, H.J. (1999) Escherichia coli O157 in farm animals, CABI Publishing, New York, USA.

36. Tree, J.J., Wolfson, E.B., Wang, D., Roe, A.J. and Gally, D.L. (2009). Controlling injection: regulation of type III secretion in enterohaemorrhagic Escherichia coli. Trends in Microbiology, 17: 370-361.

37. Van-Diemen, P.M., Dziva, F., Stevens, M.P. and Wallis, T.S. (2005). Identification of enterohemorrhagic Escherichia coli O26: H-genes required for intestinal colonization in calves. Infection and Immunity, 73: 1735-1743.

38. Yekta, M.A., Goddeeris, B., Vanrompay, D. and Cox, E. (2011). Immunization of sheep with a combination of intiminγ, EspA and EspB decreases Escherichia coli O157: H7 shedding. Veterinary Immunology and Immunopathology, 140: 42-46.

‎ 10.29252/pgr.1.1.77

‎ 20.1001.1.23831367.1393.1.1.7.2

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Sahshorpour M, Amani J, Jafari M, Salmanian A H. Expression of Recombinant Chimeric EspA-intimin Protein in Nicotiana tobaccum for Oral Vaccine Development. pgr 2014; 1 (1) :77-94
URL: http://pgr.lu.ac.ir/article-1-30-en.html

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