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目的对中华绒螯蟹组织蛋白中的变应原组分进行分析鉴定和分离提取,为研制蟹类食物过敏反应的检测试剂和脱敏制剂奠定基础。方法中华绒螯蟹粗提蛋白经SDS-PAGE分离,用蟹过敏症患者血清经Westernblot分析其变应原组分。粗提蛋白经硫酸铵分段盐析和DEAE-52离子交换层析,分离提取中华绒螯蟹的主要变应原成分,并用蟹过敏症患者血清对层析产物进行ELISA分析。结果SDS-PAGE分析显示,中华绒螯蟹粗提蛋白有20多条可辨蛋白带,相对分子质量在15700～118000之间。Westernblot分析显示,7份蟹过敏患者血清与粗提蛋白均呈阳性反应,主要有9条反应带,其中相对分子质量为35000、41000、48000和66000的4条蛋白带与7份患者血清均呈阳性反应。经DEAE-52离子交换层析分离后,变应原主要出现在30%盐析段0.2mol/L洗脱峰、50%盐析段0.2和0.4mol/L洗脱峰、70%盐析段0.2和0.3mol/L洗脱峰、90%盐析段0.2mol/L洗脱峰内。结论中华绒螯蟹组织蛋白中的相对分子质量为35000、41000、48000和66000的组分是其主要变应原。
OBJECTIVE: To analyze, identify and isolate the allergen components in the tissue protein of the Chinese mitten crab (Eriocheir sinensis) to lay a foundation for the development of detection reagents and desensitization preparations for allergic reactions to crabs. Methods The crude extracts of Eriocheir sinensis were separated by SDS-PAGE and their allergen components were analyzed by Western blot using the serum of crab allergy sufferers. The crude protein was separated by ammonium sulfate fractionation and DEAE-52 ion exchange chromatography, and the main allergen components of Eriocheir sinensis were isolated and purified. The chromatographic products were analyzed by ELISA. Results SDS-PAGE analysis showed that the crude extract of Eriocheir sinensis had more than 20 discernable protein bands with relative molecular mass between 15,700 and 118,000. Westernblot analysis showed that there were 9 reaction bands in the serum of 7 crab allergy patients and the crude protein, of which 4 bands with relative molecular mass of 35000, 41000, 48000 and 66000 and serum of 7 patients were all Positive reaction. After DEAE-52 ion exchange chromatography, the allergen mainly appeared in 0.2mol / L eluting peak in 30% salting-out section, 0.2 and 0.4mol / L eluting peak in 50% salting-out section and 70% salting-out section 0.2 and 0.3mol / L eluting peak, 90% salting-out section 0.2mol / L eluting peak. Conclusion The components of relative molecular mass of 35.00, 41000, 48000 and 66000 in Eriocheir sinensis tissue protein are their major allergens.