Cloning of Mycobacterium avium paratuberculosis hsp70 gene into pET-24a plasmid and its expression in Escherichia coli

Subject Areas : Veterinary Clinical Pathology

Rasa Sheini mehrabzade ¹ , Masoudreza Siefi abad shapori ² , Masoud Ghorbanpoor ³ , Darush Gharibi ⁴

1 - Ph.D. Student in Bacteriology, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
2 - Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
3 - Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran.
4 - Associate Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Received: 2022-04-29 Accepted : 2022-08-13 Published : 2022-11-22

Keywords: cloning, Johne's disease, Heat Shock Protein 70, Mycobacterium avium paratuberculosis, pET-24a plasmid,

Abstract :

Johne's disease (Paratuberculosis) is a disease caused by Mycobacterium avium paratuberculosis (MAP) which is a chronic and progressive infection that chiefly affects ruminants. The disease can lead to significant economic losses in the livestock industry and may also be a threat for public health; because it may be transmitted to humans through consumption of milk and other contaminated animal products. Immunity and resistance against the Johne's disease is mainly due to cell mediated immune response. Heat Shock Protein 70 (HSP70) of this bacterium is one of its important proteins, which the immune response against it can prevent the fecal excretion of bacteria. In order to facilitate the production of recombinant vaccine against Johne's disease, in this study the recombinant HSP (recombinant HSP; rHSP) was produced and its immunogenicity investigated in rabbits. For this purpose, the hsp70 gene was cloned into pET-24a plasmid and the resulting recombinant plasmid was transferred to E. coli strain BL21. The expression of the above protein was checked by SDS-PAGE and the accuracy of the nucleotide sequence was confirmed by sequencing. Immunization of rabbits by rHSP70 resulted in the production of high levels of antibodies. Based on the findings, it seems that the HSP70 specific antibody can be evaluated in the design of diagnostic methods of the disease and the produced recombinant protein can be assessed for the production of recombinant vaccines.

References:

• Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., et al. (1995). Short protocols in molecular biology. ‏ 3rd ed., New York: John Wiley & Sons, pp: 158-502.


• Bageri, S., Pourmehdi Broujeni, M., Haji Hajikolaei, M.R. and Ghorbanpoor Najafabadi, M. (2107) Seroprevalence of Mycobacterium avium subspecies paratuberculosis infection in goats of Khuzestan province. Veterinary Clinical Pathology, 10(4): 297-305. [In Persian]


• Bannantine, J.P., Rosu, V., Zanetti, S., Rocca, S., Ahmed, N. and Sechi, L.A. (2008). Antigenic profiles of recombinant proteins from Mycobacterium avium subsp. paratuberculosis in sheep with Johne's disease. Veterinary Immunology and Immunopathology, 122(1-2): 116-125.


• Colston, A., McConnell, I. and Bujdoso, R. (1994). Cloning and expression in Escherichia coli of DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis, the causative agent of Johne's disease. Microbiology, 140(12): 3329-3336.


• Constable, P.D., Hinchcliff, K.W., Done, S.H. and Grünberg, W. (2017). Veterinary Medicine, 11th ed., Missouri, Elsevier, pp: 552-565.


• Dheenadhayalan, V., Shin, K.S., Chang, C.F., Chang, C.D., Wang, S.J., McDonough, S., et al. (2002). Cloning and characterization of the genes coding for antigen 85A, 85B and 85C of Mycobacterium avium subsp. paratuberculosis. DNA Sequence, 13(5): 287-294.


• Gopal, G.J. and Kumar, A. (2013). Strategies for the production of recombinant protein in Escherichia coli. The protein Journal, 32(6): 419-425.


• Griffiths, K.L. and Khader, S.A. (2014). Novel vaccine approaches for protection against intracellular pathogens. Current Opinion in Immunology, 28: 58-63.


• Gurung, R.B., Purdie, A.C., Whittington, R.J. and Begg, D.J. (2014). Cellular and humoral immune responses in sheep vaccinated with candidate antigens MAP2698c and MAP3567 from Mycobacterium avium subspecies paratuberculosis. Frontiers in Cellular and Infection Microbiology, 4: 93.


• Koets, A., Hoek, A., Langelaar, M., Overdijk, M., Santema, W., Franken, P., et al. (2006). Mycobacterial 70 kD heat-shock protein is an effective subunit vaccine against bovine paratuberculosis. Vaccine, 24(14): 2550-2559.


• Koets, A.P., Rutten, V.P., de Boer, M., Bakker, D., Valentin-Weigand, P. and van Eden, W. (2001). Differential changes in heat shock protein­-, Lipoarabinomannan-, and purified protein derivative-specific immunoglobulin G1 and G2 isotype responses during bovine Mycobacterium avium subsp. paratuberculosis infection. Infection and Immunity, 69(3): 1492-1498.


• Koets, A.P., Rutten, V.P.M.G., Hoek, A., Bakker, D., Van Zijderveld, F., Müller, K.E., et al. (1999). Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis. Veterinary Immunology and Immunopathology, 70(1-2): 105-115.


• Langelaar, M.F.M., Hope, J.C., Rutten, V.P.M.G., Noordhuizen, J.P.T.M., Van Eden, W. and Koets, A.P. (2005). Mycobacterium avium subsp. paratuberculosis recombinant heat shock protein 70 interaction with different bovine antigen presenting cells. Scandinavian Journal of Immunology, 61(3): 242-250.


• Manning, E.J. and Collins, M.T. (2001). Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis. Revue Scientifique et Technique (International Office of Epizootics), 20(1): 133-150.


• Markey, B., Leonard, F., Archambault, M., Cullinane, A. and Maguire, D. (2013). Clinical veterinary microbiology e-book. Elsevier Health Sciences, pp: 161-176.


• Okuni, J.B., Kateete, D.P., Okee, M., Nanteza, A., Joloba, M. and Ojok, L. (2017). Application of antibodies to recombinant heat shock protein 70 in immunohistochemical diagnosis of mycobacterium avium subspecies paratuberculosis in tissues of naturally infected cattle. Irish Veterinary Journal, 70(1): 1-7.


• Rosano, G.L. and Ceccarelli, E.A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5: 172.


• Santema, W., Hensen, S., Rutten, V. and Koets, A. (2009). Heat shock protein 70 subunit vaccination against bovine paratuberculosis does not interfere with current immunodiagnostic assays for bovine tuberculosis. Vaccine, 27(17): 2312-2319.


• Santema, W., Van Kooten, P., Hoek, A., Leeflang, M., Overdijk, M., Rutten, V., et al. (2011). Hsp70 vaccination-induced antibodies recognize B cell epitopes in the cell wall of Mycobacterium avium subspecies paratuberculosis. Vaccine, 29(7): 1364-1373.


• Stills, H.F. (2012). Polyclonal antibody production. In The laboratory rabbit, Guinea pig, Hamster, and other rodents. Lexington: Academic Press,‏ pp: 259-274.


• Tolouei Kaleibar, M., Mogaddam, G. and Fahimi, M. (2016) Evaluation of clinical and intestinal ultrasonographic findings in cows with Johne's disease. Veterinary Clinical Pathology, 10(1): 11-25. [In Persian]


• Vemulapalli R., He Y., Boyle S.M., Sriranganathan N. and Schurig G.G. (2000). Brucella abortus strain RB51 as a vector for heterologous protein expression and induction of specific Th1 type immune responses. Infection and Immunity, 68(6): 3290.


• Whittington, R.J., Marshall, D.J., Nicholls, P.J., Marsh, I.B. and Reddacliff, L.A. (2004). Survival and Dormancy of Mycobacterium avium subsp. paratuberculosis in the Environment. Applied and Environmental Microbiology, 70(5): 2989-3004.


• Whittington, R.J., Marsh, I., Turner, M.J., McAllister, S., Choy, E., Eamens, G.J., et al. (1998). Rapid detection of Mycobacterium paratuberculosis in clinical samples from ruminants and in spiked environmental samples by modified BACTEC 12B radiometric culture and direct confirmation by IS 900 PCR. Journal of Clinical Microbiology, 36(3): 701-707.