Cat Allergen Recombinant Protein Mutant’s Construction And Immunological Identification

Background:Cat allergens are common indoor inhaled allergens, and participate theIgE-mediated allergic reactions all over the world universally, has been idealprotein molecule for development of new Allergen-specific immunotherapy(ASIT), that molecule plays a very important role in the regulation of allergicinflammation reaction. The IL-10is a immune inhibitory factor with variety ofbiological activity among those molecule factors, it is a protective effect for thebody. In order to reduce the allergenic of the protein recombinant, we do thecomprehensive analysis to the antigenicity of Fel d1MHC-II in the chimeric geneon the basis of Fel d1-IL-10(FIL) recombinant chimeric protein which isconstructed in the laboratory, to determine the key site of antigen reformation, anddo the transformation to the certain site with high antigen level in the cat allergenamino acid sequences, by through the site-specific mutagenesis technology ingenetic engineering, then we import the transformed gene sequences into thecarrier, transferred into the Escherichia coli to do the inducible expression. Weanalysis the mutants of recombinant chimeric protein and acquire pure target protein after the immunological identification, to lay a solid foundation for thenext step of the target protein functional test.Objective:To analysis the antigenic epitope binding of allergen Fel d1MHC-II viabioinformatics software, we ensure that the protein tertiary structure constant, toget the hypoallergenic original preparation with weakened antigenicity bytransforming the antigenicity purposively, to reduce the immune response of Feld1and induce the immune tolerance through the immunomodulatory effect ofIL-10, thereby obtain the easily standardized, safe and efficient cat allergenpreparations, to provide theoretical support and material guarantee of furtherdevelopment of allergen vaccines used in clinical treatment.Methods:1: Using online software NetMHC II2.2Server to analysis the antigenic epitopebinding of the amino acid sequence from cat allergen Fel d1, to determine thehigh value sites which are need to be transformed.2: To ensure that the protein tertiary structure unchanged and follow the principleof the antigen transformation, by using the amino acids with low polarity, lessbranched chain and aromatic nucleus to replace the amino acids with highpolarity, more branched chain and aromatic nucleus, to get the amino acidsequences with low antigenicity,3: To do the site-directed mutagenesis, inducible expression and purification ofthe target protein to the selected high-value sites using genetic engineeringmethodology；4: To do the immunological identification of each mutant recombinant protein.Results: 1: Predicted2high-value MHC-II epitopes on the cat main allergen Fel d1.2: Constructed3mutational recombinant protein on the basis of these2epitopes.Conclusion:Predicted2high-value MHC-II epitope alleles by analyzing the allergen’santigenicity of Fel d1-IL-10chimeric gene, and do the site-directed mutagenesisto these2sites, constructed3restructuring protein mutants, and laid a solidfoundation for the further functional testing and verification of the recombinantprotein mutans.