Preparation Of Murine Neutralizing Botulinum Neurotoxin B Antibodies By Using Phage Antibody Libraries And Initial Characterization On Their Immunologic Activity

Botulinum neurotoxins(BoNTs) produced by Clostridium botulinum are the most toxic substances of their class known. Human botulism is most frequently caused by types A,B and E. The dissertation is to construct a new technical method, which can preparate and select murine neutralizing botulinum neurotoxin antibodies quickly based on direct immune and phage display. And then biology activity of high affinity antibodies can be evaluated directly. So cloning gene, selecting and panning high affinity antibodies can be fulfilled in one-step with phage display, which has some advantages such as simplicity of manufacture and saving development time.To produce antibodies capable of neutralizing botulinum neurotoxin type B (BoNT/B), We cloned the carboxy-terminal end of Hc containing the major determinants responsible for specific toxin ,induced and purifed. After mice were immunized with BoNTB/Hc, the spleens were harvested ,and single-chain Fv (scFv) and Fab phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes.In the experiment, the carboxy-terminal end of He containing the major derminants responsible for specific toxin was cloned, and the identity of the cloning and Genbank sequences was up to 99%. The sequence was cloned in pET22, but the protein was not obtained. Then cloned in pET32, the fusion protein was havested. After the primers was revised, the BoNTB/Hc N-terminal modified was achieved and puried. Mice were challenged with 6×10~3 50% lethal doses of toxin to a certian extent which were vaccinated with the modified protein. At the same time, a hen was immunized with the...