中华绒螯蟹组织蛋白中变应原组分的分析与提取

Analysis and Extraction of Allergic Components in Tissue Protein of Eriocheir sinensis

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 and extract the allergic components in tissue protein of Eriocheir sinensis and provide a ba- sis for developing detection kit and desensitizer of crab allergic diseases. Methods The crude extract of tissue protein of Eriocheir sinensis was identified by SDS-PAGE and analyzed for allergic components by Western blot, from which the major allergic components were separated by fractional ammonium sulfate precipitation and DEAE-52 ion exchange chromatography and analyzed by ELISA using the sera of patients with crab allergic diseases. Results The crude extract of tissue protein of Eriocheir sinensis showed about 20 bands, with relative molecular masses of 15 700 ~ 118 000, on SDS-PAGE profile. Western blot showed positive reactions of the crude extract with all the 7 serum samples from patients with crab allergic diseases. Nine protein bands of crude extract showed positive reactions with most of the serum samples, of which 4 （Mr 35 000, 41 000, 48 000 and 66 000） showed positive reactions with all the 7 samples. After separation by DEAE-52 ion exchange chromatography, ELISA proved that the allergic components mainly existed in the peaks of 30% ammonium sulfate precipitation eluted with 0.2 mol/L sodium chloride, 50% ammonium sulfate precipitation eluted with 0. 2 and 0. 4 mol/L sodium chloride, 70% ammonium sulfate precipitation eluted with 0. 2 and O. 3 mol/L sodium chloride and 90% ammonium sulfate precipitation eluted with 0. 2 mol/L sodium chloride. Conclusion The components with relative molecular masses of 35 000, 41 000, 48 000 and 66 000 were major allergens in tissue protein of Eriocheir sinensis.