Staphylococcus aureus (S.aureus) is the most common pathogen found in hospitals, community-acquired infections and colonizes up to 50% of humans, including mucous membranes and damaged skin. Some strains produce enterotoxins (Ent) as staphylococcal enterotoxin A (SEA) which is involved in 75% of food poisoning outbreaks. Few methods are sensitive and specific enough to confirm the diagnosis of staphylococcal food poisoning. In this study, a segment of Ent A-ORF with molecular weight 774 bp was amplified, cloned, sequenced and aligned with published Ent A-ORFs. Ent A-ORF was subcloned into bacterial expression vector (GST-PGEX4T1 vector) and expressed as a fusion protein with GST-tagged protein. A band size of 27 kDa of purified Ent A protein was cleaved from GST protein by thrombin. The expressed protein of Ent A was identified by strong reaction with commercialized polyclonal antibodies against Ent A. Antiserum against Egyptian recombinant Ent A protein was produced by immunization of Balb/c mice, the produced recombinant polyclonal antibodies had a titre of 1:2500 in direct ELISA. The produced recombinant polyclonal antibody was evaluated and reacted specifically in Western immunoblotting analysis and ELISA test. Finally from the obtained results, the produced recombinant protein of Ent A can be used successfully for production of recombinant polyclonal antibodies and used in large-scale detection of staphylococcal enterotoxin A.
| [1] | Agrawal, R., Singh, P.K., Sharma, S.K., Kamboj, D.V., Goel, A.K., & Singh, L. (2012). “Highly Expressed Recombinant SEB for Antibody Production and Development of Immunodetection System,” Indian J. Microbiol., 52(2): 191-196.View Article PubMed |
| [2] | Rasooly, R., Do, P., & Hernlem., B. (2016). “Sensitive, Rapid, Quantitative and in Vitro Method for the Detection of Biologically Active Staphylococcal Enterotoxin Type E. Toxins,” 8, 150.View Article PubMed |
| [3] | Wiedmann, M. & Zhang, W. (2013). “Genomics of Foodborne Bacterial Pathogens,” Springer New York Dordrecht Heidelberg London. ISBN 978-1-4419-7685-7.View Article |
| [4] | Pinchuk, I.V., Beswick, E.J. & Reyes, V.E. (2010). “Staphylococcal enterotoxins,” Toxins, (Basel), 2: 2177-2197.View Article PubMed |
| [5] | Le Loir Y, Baron F, Gautier M. (2003). “Staphylococcus aureus and food poisoning,” Genet. Mol. Res, 2: 63-76. PubMed |
| [6] | Rall, V.L.M., Sforcin, J.M., Augustini, V.C.M., Watanabe, M.T., Fernandes Jr., A., Rall, R., Silva, M.G., & Araújo, Jr.J.P. (2010). “Detection of enterotoxin genes of Staphylococcus sp. Isolated from nasal cavities and hands of food handlers,” Braz. J. Microbiol., 41: 59-65.View Article PubMed |
| [7] | Hussein, H.A., Ibrahim, M.K., Nour El-Din, H.A., El-Shatoury, E.H., El-Fouly, M. E., Abd-Eal, S.S. & Abu El-Naga, M.N. (2016). “Molecular Screening of Staphylococcal Enterotoxin Type A Encoding Gene from MRS Clinical Isolates,” American Journal of Microbiological Research, 4 (2): 68-72. View Article |
| [8] | Chen, L., Li, S., Wang, Z., Chang, R., Su, J. & Han, B. (2012). “Protective effect of recombinant staphylococcal enterotoxin A entrapped in polylactic-co-glycolic acid microspheres against S. aureus infection,” Veterinary Research, 43(1): 20. Available at: http://www.veterinaryresearch.org/content/43/1/20.View Article PubMed |
| [9] | Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989). “Molecular Cloning”: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor New York. |
| [10] | Hammond, J. & R. W. Hammond (1989). “Molecular cloning, sequencing and expression in Escherichia coli of the bean yellow mosaic virus coat protein gene. Journal of General Virology 70:1961-1974.View Article PubMed |
| [11] | Hanahan, D. & M. Meselson (1983). “In Methods in Enzymology,” Vol. 100, Wu. et al. (eds), Acad. Press. NY, p. 333. |
| [12] | Altschul, S.F., Boguski, M.S., Gish, W. & Wootton, J.C. (1994). “Issues in searching molecular sequence databases,” Nat Genet. 6(2):119-29.View Article PubMed |
| [13] | Krzywinski, M., Schein, J., Birol, I., Connors, J., Gascoyne, R., Horsman, D., Jones, S.J. and Marra, M.A. (2009). “Circos: an information aesthetic for comparative genomics,” Genome Res., 19(9):1639-45.View Article PubMed |
| [14] | Kelley, L.A. & Sternberg, M.J E. (2009). Protein structure prediction on the web: a case study using the Phyre2 server,” Journal of Nature Protocols, 4: 363-371.View Article PubMed |
| [15] | Marco, B., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Cassarino, T. G., Bertoni, M., Bordoli, L. & Schwede, T. (2014). “SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information,” Nucleic Acids Res., 42 (1): 252-258.View Article |
| [16] | Towbin H, Staehelin T, & Gordon J. (1979). “Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications,” Proc. Natl. Acad. Sci. USA, 76(9): 4350-4.View Article PubMed |
| [17] | Kuang, H., Wang, W., Xu, L., Ma, W., Liu, L., Wang, L. & Xu, C. (2013). “Monoclonal antibody-based sandwich ELISA for the detection of staphylococcal enterotoxin A,” Int. J. Environ. Res. Public Health, 10:(4) 1598-608.View Article PubMed |
| [18] | Avrameas, S. (1969). “Coupling of enzymes to proteins with glutaraldehyde.” Immunochemistry, 6: 43-52.View Article |
| [19] | Clark, M.F. & A.N. Adams (1977). “Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses, ” J. Gen. Virol., 34: 475-483.View Article PubMed |
| [20] | McLaughlin, M.R., Barnett, O.W., Gibson, P.B. & Burrows, P.M. (1984). “Enzyme linked immunosorbent assay of viruses infecting forage legumes, ” Phytopathology 74: 965-969.View Article |
| [21] | Betley, M.J. & Mekalanos. J.J. (1988). “Nucleotide sequence of the type A staphylococcal enterotoxin gene,” J. Bacteriol. 170(1): 34-41.View Article PubMed |
| [22] | Weir, D., Jones, C., Ammerman, L., Dybdahl, K. & Tomlinson, S. (2007). “Report of a strain of Staphylococcus caprae with the genes for enterotoxin A and enterotoxin-like toxin type P,” J. Clin. Microbiol., 45 (10): 3476-3477.View Article PubMed |
