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.
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 L1 (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.
Kumar
T D Kalyan
TDK
Division of Microbiology, Defense Food Research Laboratory, Siddhartha Nagar, Mysore, Karnataka 570011, India.
Balakrishna
K
K
Division of Microbiology, Defense Food Research Laboratory, Siddhartha Nagar, Mysore, Karnataka 570011, India.
Murali
H S
HS
Division of Microbiology, Defense Food Research Laboratory, Siddhartha Nagar, Mysore, Karnataka 570011, India.
Batra
H V
HV
Division of Microbiology, Defense Food Research Laboratory, Siddhartha Nagar, Mysore, Karnataka 570011, India.
D001409:Bacillus cereus / Q000235:genetics*; D001413:Bacillus thuringiensis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D017931:DNA Primers; D004768:Enterotoxins / Q000235:genetics*; D005517:Foodborne Diseases / Q000235:genetics / Q000382:microbiology*; D006360:Heat-Shock Proteins / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics*; D006801:Humans; D008089:Listeria monocytogenes / Q000235:genetics* / Q000302:isolation & purification; D008088:Listeriosis / Q000235:genetics*; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000235:genetics*; D012275:Oryza / Q000382:microbiology; D010957:Plasmids; D013203:Staphylococcal Infections / Q000235:genetics*; D013211:Staphylococcus aureus / Q000235:genetics*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004768:Enterotoxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D050506:Mutant Chimeric Proteins; C083961:hemolysin BL protein, Bacillus; C031818:enterotoxin B, staphylococcal; C038705:hlyA protein, Listeria monocytogenes