腐食酪螨过敏原的分析鉴定与纯化

Identification and Purification of Tyrophagus putrescentiae Allergens

目的分析、鉴定与纯化腐食酪螨过敏原组分。方法以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析鉴定腐食酪螨蛋白质提取液中的蛋白质组分和过敏原组分,并通过阴离子交换层析和分子排阻色谱进一步纯化。结果腐食酪螨的蛋白质粗浸液电泳后可以辨认的蛋白质带共23条,相对分子质量(Mr)分别为177000、118000、107000、70000、67000、60000、52000、45000、41000、40000、38000、37000、35000、27000、23000、22000、18000、17000、16000、15000、14000、13000和12000。Westernblotting分析显示,腐食酪螨的5条致敏蛋白质带分别为Mr128000、67000～70000、36000～37000、18000和16000,其中前3条带特异性结合IgE阳性率均为100%,而Mr18000和Mr16000条带的阳性反应率分别为77.8%和44.44%。通过阴离子交换层析和分子排阻色谱从腐食酪螨粗浸液中分离到Mr18000的过敏原组分。结论腐食酪螨有4种主要过敏原及1种次要过敏原。

Objective To identify and purify the allergens of Tyrophagus putrescentiae. Methods Tyrophagus putrescentiae extract was analyzed by SDS-PAGE and Western blotting, and was partially purified by DE52 anionexchange chromatography and HiLoad 16, 60 Superdex 200 prep grade Size-exclusion chromatography. Results 23 bands were found after SDS-PAGE with comparative molecular weight （Mr） of 177 000, 118 000, 107 000, 70 000, 67000, 60000, 52000, 45000, 41000, 40000, 38000, 37000, 35000, 27000, 23000, 22000, 18000, 17000, 16 000, 15 000, 14 000, 13 00 and 12 000. Five allergens were detected by Western blotting with Mr 128 000, 67 000- 70 000, 36000-37000, 18000 and 16000, respectively. The positive reaction rate of 3 allergens, with Mr 128 000, 67 000- 70 000 and 36 000-37 000, were 100%, while that of other 2 allergens with Mr 18 000 and 16 000 was 77.8% and 44.4% respec tivelyo The allergen with Mr 18 000 was purified by anion-exchange chromatography and Size-exclusion chromatography. Conclusion Four major allergens and one minor allergen from Tyrophagus putrescentiae have been identified.