| Research Abstract |
Several glycoproteins with asparagine-linked sugar chains were expressed in insect cells, and the glycoproteins with fucose-containing sugar chains were produced. First, the cDNA encoding each protein was inserted into a transfer vector to introduce into baculovirus genome DNA. The viral genome DNA and the transfer vector containing the CDNA were introduced into a insect cell line, BTI TN 5B1-4. The virus particles were recovered from the culture supernatant, and its betagalactosidase activity was measured. The galactosidase-positive recombinant virus was selected and cloned. Each recombinant virus was cultured in a larger scale, and recombinant glycoproteins expressed in the insect cells were purified. To determine whether sugar chains with the carbohydrate epitopes were added to the recombinant proteins, the purified glycoproteins were subjected to immunoblotting and ELISA using the antiserum specific for the carbohydrate epitope. Both of the blot and ELISA demonstrated that all the recombinant glycoproteins tested were clearly positive to the carbohydrate epitope-specific antibody, though the reactivity varied from one protein and another. Furthermore, the presence of the carbohydrate epitope was confirmed from the observation that the antigenic reactivity of each recombinant glycoprotein was lost by the periodate treatment. Further detailed analyzes to characterize the carbohydrate structure are in progress including lectin-binding assay, glycosidase treatments, chemical analysis, and immunochemical analyzes using patient's antibodies. The present research demonstrated that recombinant glycoprotein allergens with carbohydrate epitopes can be produced in the baculovirus/insect cell expression system, and suggests that the recombinant proteins show allergenic reactivity comparable to natural allergens. Thus, baculovirus/insect cell expression system would be a useful tool for theproduction of glycoprotein allergens for research and diagnosis purposes.
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