Abstract: To analyse, identify and primarily purify Bombyx mori specific allergen using mass spectrometry, we made the crude extracts of different growing stages of the silkworm by Coca's extracts, identified its specific allergens by SDS-PAGE and Western blotting, purified the 30 kD specific allergenby DEAE-52 ion exchange chromatography and gel isolation, and analysed the data through website-searching after MALDI-TOF-MS online analysis. The results indicated that the silkworms at different growing stages had about 20 protein bands, and the 5th instar larvae had 23 protein bands. The molecular weights of the 11 major bands were 82, 79, 60, 51, 46, 38, 32, 30, 28, 24 and 18 kD, respectively. The results of Western blotting with the positive serum of anaphylactic patients showed that the 1st－4th instar larvae all had the specific allergens whose molecular weights were 82 and 79 kD, respectively; only the 5th instar larvae all had the specific allergen of 30 kD. We purified the protein of 30 kD by ion exchange chromatography and gel isolation and identified it as ectoblast protein by MALDI-TOF-MS. The reults suggest that the antigen changes with different stages and the protein of 30 kD appeared newly at the 5th instar larva is stage-specific.

Key words: Bombyx mori, allergen, SDS-PAGE, Western blotting, ion-exchange chromatography, mass spectrometry