Construction And Expression Of Pea Based Immunotoxin With Antigenicity Attenuated

The issue of using human Fc fragment as a targeting part of the chimeric IgE Pseudomonas aeruginosa exotoxin A (PEA), building a targeted toxin. Then using bioinformatics software to analyze the MHC type II antigenicity of targeted toxins, evaluated the results of several software and determine the highest value of the antigen sites. By site-directed mutagenesis technology to change its nucleic acid sequence , the purpose is reducing its antigenicity. And then transformed the plasmid into E. coli, induce its formation the target protein, induced to produce the target protein. Using nickel column affinity to purify target protein based on AKTA AFLP system, and then dialysis makes the protein refolding, while removal the ion composition, the target protein was isolated and purified finally, for the next step lay a solid foundation.Object:Fc fragment of human IgE as a target part, chimeric Pseudomonas aeruginosa exotoxin A (PEA) to construct targeted toxin, and analyze its MHC-II type antigenicity, transform their antigens sites with high value, obtain the protein with antigenicty attenuation. Methods:1. The coding sequences of PEA and the Fc fragment of human IgE were combined and the codons of whole sequence were optimized for protein expression in E. coli.2. The power of their antigenicity prediction of several bioinformatic softwares was evaluated by using well-studied peptides as common materials to be predicted.3. The antigenicity of the chimeric protein constructed relating to MHC-â…¡molecules were predicted by bioinformatic software. And the amino acide sites with high antigenicty were resolved.4. Two amino acid sites were respectively replaced by site-directed mutagenesis kit.5. The mutated coding genes were respectively expressed in the E. coli and the expressed proteins were purified by nickel affinity chromatography.Results:1. The codons of chimeric gene were optimized and expression construct were obtained.2. Three plasmids with different mutation sites were acquired.3. Four chimeric proteins were expressed and purified, including the untouched toxin, two of the recombinant proteins each with a single mutation site, the recombinant protein with two mutation sites.Conclusion:The theoretic antigenicity of the PEA based immunotoxins was attenuated. Four homology proteins with different antigenicities were expressed and purified, which would lay a solid foundation for furthur experiment.