Production and characterization of a recombinant chimeric antigen consisting botulinum neurotoxin serotypes A, B and E binding subdomains.
Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this study, polypeptides from C-terminal heavy chain of BoNTs serotypes A, B and E to the length of 54, 45 and 48 amino acid respectively were selected, linked together using a hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30 degrees C and 37 degrees C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody titer as well as the extent of protectivity were determined. Mice given three injections (10 microg/mice) of the antigen were protected against an intra-peritoneal administration of 10 LD(50 )of serotypes A and E, but 100 LD(50) of serotype B. We conclude that a significant correlation exists between the antigenic characteristics and protection capability of the chimeric protein prepared in this study.
Ebrahimi
Firouz
F
Department of Clinical Biochemistry, Medical Sciences Faculty, Tarbiat Modares University, Tehran, I.R. Iran.
Rasaee
Mohammad Javad
MJ
Mousavi
Seyed Latif
SL
Babaeipour
Valiollah
V
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005732:Gangliosides / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D050861:Synaptotagmin II / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014611:Vaccination
D005732:Gangliosides; D011993:Recombinant Fusion Proteins; D050861:Synaptotagmin II; C016481:trisialoganglioside GT1; D001905:Botulinum Toxins