Research Of Genetically Engineered Antibody Against BoNT/B Based On Epitope Prediction

The botulinum neurotoxins (BoNTs) produced by anaerobic Clostridium botulinum are the most potent and lethal toxins known to man. Mature toxin is composed of the light chain and heavy chain linked by a disulphide bond. The present research had identified the heavy chain of BoNT could induced the organism to produce protective neutralized antibody, while the epitope location is still unknown. The objective of this research was to predict the epitope with the help of bioinformation, and trying to establish a technical routine to obtain the epitope specific antibody based on prediction, which will provide a new way for research of genetically engineered antibody against BoNT/BFirst, with the help of bioinformatics, according to the amino acid structure of BoNT/B heavy chain and the utilization of Anthwine, Goldkey software, 8 epitope peptides were synthesized with the considering of hydrophilicity, accessibility, flexibility, and secondary structure multi parameters. The immune test in vivo and in vitro indicated the peptides were of good antigenicity.Then, on the base of epitope prediction, BALB/C mice were immunized with the epitope peptides, spleen cells were harvested, the total antibody variable region genes were cloned, and spliced by SOE( sequence overlap extension) in the way of VH-linker-VL. A 1.6x10 phage displayed ScFv( single chain variable fragment of antibody )immuned library was constructed after 20 times electrotransformation. After 3-4 cycles of screening against the epitope peptide targets, phage displayed ScFv were enriched and specific binders were identified by competition-inhibition assay. What ever, the ScFv-P6-12, ScFv-P7-8, ScFv-P8-12 against P6, P7, P8 individually could...