Methods: The fusion molecule was generated by molecular cloning and expressed in E. coli. Receptor binding studies were performed by immunoblot, ELISA, and flow cytometry. Activation of basophils was studied by basophil activation test (BAT) with blood from shrimp allergic individuals.
Results: Tropomyosin and the C-terminal part of fibroleukin were fused by a short flexible linker consisting of the amino acids RADAAP. The fusion protein bound to the human Fcgamma-receptor-IIb in immunoblot and ELISA and binding of the fusion protein to human B-cells was shown by flow-cytometry. Shortening of the allergen into a peptide covering one-fifth of whole tropomyosin increased the binding to B-cells. Futhermore, a decrease in the activation of basophils to shrimp tropomyosin was observed in presence of the fusion protein.
Conclusions: Here we describe a novel fusion protein based on fibroleukin and shrimp tropomyosin that may have tolerizing effects on basophils and B-cells in shrimp allergic individuals.