家蚕30 kD特异性变应原的分析、纯化与质谱鉴定

以Coca's提取液分别提取到不同时期家蚕Bombyx mori的粗浸液，利用SDS-PAGE和Western blotting鉴定其特异性变应原，然后用DEAE-52离子交换层析及切胶纯化出30 kD的特异性变应原，再经MALDI-TOF在线联机分析，所得质谱数据进入网站搜索分析。结果显示：1～5龄家蚕均有20条左右蛋白带，其中 5龄家蚕有23条蛋白带，主带有11条（82、79、60、51、46、38、32、30、28、24和18 kD）。选用家蚕过敏患者阳性血清进行免疫印迹，1～4龄家蚕均显示出82和79 kD的特异性变应原；但只有5龄家蚕的30 kD蛋白为特异性变应原，通过离子交换层析和经切胶纯化出30 kD蛋白，再经MALDI-TOF-MS鉴定该蛋白为外膜蛋白。提示家蚕不同时期抗原成分有所变化，5龄家蚕新出现的30 kD蛋白为特异性变应原。

Analysis, purification and identification of the 30 kD specific allergen of Bombyx mori using mass spectrometry

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.