Preliminary Identification Of Neutralizing Epitopes Of Botulinum Neurotoxin Type A By Ribosome Display Technology

In this experiment, to express, purify and analyze the antigenicity of the heavy chain C-terminal binding domain of botulinum neurotoxin type A(BoNT/AHc-C), the gene encoding BoNT/AHc-C was optimized by replacing rare codons with high-frequency ones and adjusting AT contents, then synthesized using overlapping PCR. The synthetic gene was subcloned into the pET-His vector for expression in Escherichia coli, and recombinant protein was one-step purified by using Ni-NTA affinity agarose chromatography column, then refolded. The antigenicity of recombinant BoNT/AHc-C was identified by western blot and indirect ELISA. The rBoNT/AHc-C, which obtained a 65 % overexpression in the genetically engineering strain BL21/pET-BoNT/AHc-C, was one-step purified and refolded to 95% purity.To prepare the monoclonal antibodies (mAbs) against rBoNT/AHc-C, BALB/c mice were intraperitoneally immunized with purified rBoNT/AHc-C, and splenocytes of immunized mice were fused with myeloma cells Sp2/0. Hybridoma cells were screened by indirect ELISA and limited dilution was used to obtain mAbs. Three hybridomas cell lines producing the mAbs against BoNT/A, named 2A8 4F7 and 2F2, were successfully established. Identification of subclass showed that all mAbs belong to IgGl These mAbs showed high specificity to recombinant BoNT/A-Hc and nativeBoNT/A by the indirect ELISA. The mAb 2A8 showed preferable neutralizing activity.To obtain all elements used for ribosome display, plasmid pT7PD was constructed by Overlapping PCR, which can be transcribed and translated in vitro efficiently. Then, a high-complexity DNA library which encodes random 12-mers peptide used for prokaryotic ribosome display was generated by PCR and DNA ligation. The DNA library was identified by sequencing and in vitro transcription/translation. Ribosome display was applied to select the immobilized mAb 2A8 and positive result was obtained. This paves the way for epitope mapping for monoclonal antibodies by ribosome display technology.