Construction Of Mast Cell-Targeted Recombinant Immunotoxins For Antigenicity Attenuation

Background: Mast cells around cancer tissue and a variety of cytokines produced by mast cells play an important role in new blood vessel formation around tumor cells and promote the growth and development of cancer. Removal of these mast cells maybe an important approach on treatment of cancer. We analyzed its MHC-â… antigenicity, and determined two key sites in Ig-Fc-PEA and mutated these sites with some amino acids to eliminate MHC-â… antigenicity. These chimeric immunotoxins were expressed in E.coli, and purified by nickel column affinity, refolded by dialysis to removel ion compositions. This study provides a solid foundation for further function validation of these chimeric immunotoxins and would offer a novel approach on the treatment of cancer.Objective:We purpose to construct immunotoxins with antigenicity attenuation through analyzing its MHC-â… epitopes based on Ig-Fc-PEA and get less allergenicity immunotoxins in order to enhance clinical treatment and reduce related side effect. Methods:1. MHC-â… antigen epitopes of PEA amino acid sequence were analyzed using the MHC-â… antigen epitope predication software SYFPEITHI to determine the sites of modification required;2. The amino acids with high polarity, branched chain and aromatic nucleus were replaced to get the amino acid sequences with low antigenicity;3. These amino acid sites were respectively replaced by site-directed mutagenesis kit, expressed in the E. coli and purified by nickel affinity chromatography;4. These immunotoxins were identified by Western blotting.Results:1. Two important MHC-â… epitope sites in PEA were obtained;2. On this basis of the two epitopes on the recombinant, we expressed six important mutatated proteins.Conclusion:We constructed and expressed six modified immunotoxins based on two important MHC-â… epitope sites on Ig-Fc-PEA immunotoxin, which laid a solid foundation for follow function and other tests.