title: B-cell epitope of beta toxin of Clostridium perfringens genetically conjugated to a carrier protein: expression, purification and characterization of the chimeric protein.
abstract: Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain, known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines targeting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic load in the host leading to nonspecific and poor responses. The present study aimed to overcome these drawbacks and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrier protein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted as putative B-cell epitopes. One of the epitopes spanning 140-156 amino acid residues was genetically conjugated with B-subunit of heat labile enterotoxin (LTB) of Escherichia coli and expressed as a translational fusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusion protein rLTB-Btx140-156 was obtained and the protein was successfully purified. The recombinant protein retained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The antigenicity of both the epitope and the carrier protein was maintained in fusion protein as indicated by immunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140-156 fusion protein therefore can be evaluated as a potential vaccine candidate against C. perfringens.
authors.affiliation: Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Bharti
authors.initials: B
authors.lastname: Bhatia
authors.affiliation: Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Solanki
authors.affiliation: Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Himani
authors.initials: H
authors.lastname: Kaushik
authors.affiliation: School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India. Electronic address: adixit7@gmail.com.
authors.forename: Aparna
authors.initials: A
authors.lastname: Dixit
authors.affiliation: Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India. Electronic address: lalit@nii.ac.in.
authors.forename: Lalit C
authors.initials: LC
authors.lastname: Garg
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification; D001428:Bacterial Vaccines / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003016:Clostridium perfringens / Q000235:genetics*; D004768:Enterotoxins / Q000235:genetics* / Q000302:isolation & purification; D018985:Epitopes, B-Lymphocyte / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D014734:Vibrio cholerae / Q000235:genetics*
chemical_list: D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D018985:Epitopes, B-Lymphocyte; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C027538:heat-labile enterotoxin, E coli
keywords: Beta toxin; Clostridium perfringens; Epitope; Epitope prediction algorithms; Fusion protein; LTB; Vaccine