IgE-mediated allergic reactions are the most common hypersensitivity disorders affecting up to 30% of the population worldwide and still rising. Due to the high prevalence of these diseases, accurate diagnosis is critical to ensure the most effective treatment. Immunological test systems for allergy diagnostics currently mostly involve allergen extracts, which are a heterogeneous mixture of various allergic and non-allergic components. Levels of the main allergen and the additional substances could vary among different batches of the product. In contrast, recombinant allergens can be produced as defined molecules with known immunologic and biological features [1]. The usage of purified recombinant allergens instead of allergen extracts could increase the accuracy of allergic reaction detection, as well as reduce cross-reactions. Recombinant proteins could be produced using various expression systems, including mammalian cells. The ability to perform post-translational modifications that are not present in proteins produced in prokaryotes makes mammalian cells expression system an advantageous production platform [2].
This study aimed to produce recombinant secreted maltose-binding protein (MBP) fused wasp's allergen Ves v 2 (rVes v 2-MBP) and cow's milk allergen Bos d 4 (rBos d 4-MBP) in mammalian cells. Synthetic DNA fragments coding these allergens were cloned to mammalian expression vector and transiently transfected into Chinese hamster ovary (CHO) cells. Secreted rBos d 4-MBP was purified using MBP affinity tag and confirmed using immunoblotting and indirect enzyme-linked immunosorbent assay using monoclonal antibodies against MBP. However, recombinant Ves v 2 allergen was not secreted into cells growth media but was detected in intracellular compartment of the cell. To determine if synthesized recombinant protein antigenicity is similar to natural allergen, blood serum samples from patients allergic to cow's milk were tested with rBos d 4-MBP. Also, blood serum samples were tested using the same recombinant allergen produced in E. coli.
Results show that serum samples with allergen-specific IgE recognizes rBos d 4-MBP antigen produced in CHO cells, suggesting that recombinant protein contains IgE-binding epitopes. Nevertheless, rBos d 4-MBP protein produced in E. coli was not recognized by specific IgE.
In conclusion, recombinant Bos d 4 fused with MBP expressed in mammalian cells is most likely to be similar to native protein as compared to the same recombinant allergen produced in E. coli. These results indicate that mammalian cells expression system is suitable for eukaryotic protein production and synthesized recombinant proteins could be used for improving molecular allergology tests in vitro.