title: Construction of a non toxic chimeric protein (L1-L2-B) of Haemolysin BL from Bacillus cereus and its application in HBL toxin detection.
abstract: Among the many potential virulence factors of B. cereus, Haemolysin BL is a unique and potent three component pore forming toxin composed of a binding component, B, and two lytic components, L(1) and L(2). Heterogeneity in nucleic acid and protein sequences of HBL components and problems during expression of L(1) and L(2) proteins in recombinant host due to their toxicity causes problems for development of specific detection systems based on PCR and Immunoassay, respectively. Commercially available kit (BCET RPLA, Oxoid) is useful for detection of L(2) component of HBL, but detection of only one component is insufficient to give comprehensive view on HBL toxin producing strains as some strains produced only one or two of the three HBL components. To address above mentioned problems, in this study, we cloned conserved domains of B, L(1) and L(2) components together as single fusion gene and expressed as recombinant multidomain chimeric protein in E. coli. The resultant protein having L(1), B and L(2) components in the form of single protein had no toxicity towards E. coli as we followed truncated protein approach. The hyperimmune antisera raised in mice against r-chimeric protein reacted with all the three components of HBL toxin of B. cereus (ATCC 14579) and provided three reaction bands at ~40 kDa to ~50 kDa regions during Western blot analysis. The hyperimmune sera of r-chimeric protein also notably neutralized the hemolytic activity of native HBL toxin. These results demonstrated that the obtained chimeric protein is correct and retained the antigenicity of native HBL toxin components. Therefore, it has better application in the development of a comprehensive HBL detection immunoassay and may also be a potential candidate molecule for vaccine studies.
authors: Division of Microbiology, Defense Food Research Laboratory, Siddhartha Nagar, Mysore, Karnataka, India. devikalyan@gmail.com
authors: T D Kalyan
authors: TD
authors: Kumar
authors: H S
authors: HS
authors: Murali
authors: H V
authors: HV
authors: Batra
mesh_terms: D000818:Animals; D001409:Bacillus cereus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins