Construction of a recombinant intergenus multidomain chimeric protein for simultaneous expression of haemolysin BL of Bacillus cereus, listeriolysin O of Listeria monocytogenes and enterotoxin B of Staphylococcus aureus

• Published in: Journal of medical microbiology Vol. 58; no. 5; pp. 577 - 583
• Main Authors: Kumar, T. D. Kalyan, Balakrishna, K, Murali, H. S, Batra, H. V
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.05.2009; Society for General Microbiology
• Subjects: Bacillus cereus; Bacillus cereus - genetics; Bacillus thuringiensis - genetics; Bacterial Proteins - genetics; Bacterial Toxins - genetics; Bacteriology; Biological and medical sciences; DNA Primers; Enterotoxins - genetics; Escherichia coli; Foodborne Diseases - genetics; Foodborne Diseases - microbiology; Fundamental and applied biological sciences. Psychology; Heat-Shock Proteins - genetics; Hemolysin Proteins - genetics; Humans; Infectious diseases; Listeria monocytogenes; Listeria monocytogenes - genetics; Listeria monocytogenes - isolation & purification; Listeriosis - genetics; Medical sciences; Microbiology; Miscellaneous; Molecular Sequence Data; Mutant Chimeric Proteins - genetics; Oryza - microbiology; Plasmids; Staphylococcal Infections - genetics; Staphylococcus aureus; Staphylococcus aureus - genetics; Microbiology; Listeria monocytogenes; Bacillaceae; Toxin; Bacillus cereus; Bacillales; Gram positive bacteria; Enterotoxin; Bacteria; Micrococcales; Micrococcaceae; Recombinant protein; Hemolysin; Staphylococcus aureus
• ISSN: 0022-2615; 1473-5644
• DOI: 10.1099/jmm.0.007658-0

Division of Microbiology, Defense Food Research Laboratory, Siddhartha Nagar, Mysore, Karnataka 570011, India Correspondence T. D. Kalyan Kumar devikalyan{at}gmail.com Received October 30, 2008 Accepted January 6, 2009 Haemolysin BL (HBL) of Bacillus cereus , listeriolysin O (LLO) of Listeria monocytogenes and enterotoxin B (SEB) of Staphylococcus aureus are among the major toxin components contributing to the pathogenicity of these organisms in foodborne illnesses. In this study, an intergenus non-toxic multidomain fusion protein (r-HLE) was generated with specificity for HBL, LLO and SEB. The fusion gene ( r-hle ) comprising the conserved regions of hblD and the hly and entB genes was codon-optimized for expression in Escherichia coli and encoded a 50 kDa recombinant multidomain chimeric protein (r-HLE). Hyperimmune antiserum raised against r-HLE specifically reacted with the L 1 (38 kDa) component of the HBL complex of B. cereus , LLO (58 kDa) of L. monocytogenes and SEB (28 kDa) of S. aureus during Western blot analysis when tested on standard strains. During testing on isolates, the antiserum again identified the appropriate toxin molecules and was highly specific to the relevant bacterial species. The antigenicity of the SEB component of the r-HLE protein was also confirmed using a commercially available TECRA kit. The described procedure of creating a single antigenic molecule carrying components of three different toxins whilst still retaining the original antigenic determinants of individual toxins will be highly advantageous in the development of rapid, reliable and cost-effective immunoassays. Abbreviations: HBL, haemolysin BL; LLO, listeriolysin O; SEB, enterotoxin B. The GenBank/EMBL/DDBJ accession number for the sequence of the r-HLE protein constructed in this study is EU912538.