| Objective: The major horse allergen Equ c1is a lipocalin protein, it can induces anIgE-mediated type I allergic reaction in a majority of the patients allergic to horses. This articleaims to explore the reorganization of low allergenicity allergens Equ c1to lay the foundation forits application in specific immunotherapy. Methods: in this study, the mature peptide of Equ c1by codon optimization section was recombined expression in Escherichia coli, and theoptimization of expression conditions, affinity purification and identification by Western-Blotwere carried out. Besides, we used MHCⅡ Service2.0software to predict T-cell allergenepitopes of Equ c1, identify the three highest allergenic sites, using site-directed mutagenesis tochange specific amino acid codon, then expressed in E. coli to get the low epitope protein, thesimulation analysis of its tertiary structure, and Western-Blot identification was also carried out.Results: Rosetta-Equ c1reorganized strain can achieve optimal protein expression effect byadding0.5μL IPTG on the concentration of1mmol/L,37℃induced for6h. SDS-PAGE resultsof the recombinant Equ c1protein showed that the target band was in the position of22KDa,pure target protein was in eluting peak and elution peak. By Single locus mutation andcombinations mutations of the three units, we expressed one recombinant Equ c1protein andseven kinds of mutations Equ c1protein totally. Conclusion: after the transformation, thebinding force of the50,120,153first three antigens sliding peptide and MHC class II moleculeswere reduced to below0.42, achieving our expected results. The simulation results of the tertiarystructures show that the protein tertiary structure did not change significantly before and after thepoint mutation. The StrepII tag Western-Blot analysis showed that both the purified recombinantprotein and the mutant proteins are target protein. |
