刀额新对虾组织蛋白中变应原组分的分析与纯化

Analysis and Purification of Allergic Components in Tissue Protein of Metapenaeus ensis

目的分析和纯化刀额新对虾组织蛋白中的变应原组分,为进一步研制虾源性过敏反应疾病的诊断试剂和脱敏药物奠定基础。方法刀额新对虾粗提蛋白经SDS-PAGE分离,用虾过敏患者血清,经Western blot分析其变应原组分。以DEAE-52离子交换层析纯化主要变应原组分,用虾过敏患者血清对纯化产物进行ELISA分析。结果SDS-PAGE分析显示,刀额新对虾粗提蛋白约有20条可辨蛋白带,相对分子质量在12100～105600之间。Western blot分析显示,12份虾过敏患者血清与粗提蛋白均呈阳性反应。粗提蛋白有7条蛋白条带反应率高,其中相对分子质量为36000、46200、60000和68000的4条蛋白带与所有12份患者血清均呈阳性反应。经DEAE-52离子交换层析纯化后,ELISA结果显示,变应原主要集中在70%盐析段0.1mol/L洗脱峰内。结论刀额新对虾组织蛋白中的相对分子质量为36000、46200、60000和68000的组分是其主要变应原。

Objective To analyze and purify the allergic components in tissue protein of Metapenaeus ensis and provide basis for further development of diagnostic kit and desensitizer of shrimp allergic diseases. Methods The crude extract of tissue protein of Metapenaeus ensis was identified by SDS-PAGE and analyzed for allergic components by Western blot against the sera of patients with shrimp allergic diseases. The major allergic components were purified by fractional ammonium sulfate precipitation and DEAE-52 ion exchange chromatography, then analyzed by ELISA using the sera of patients with shrimp allergic diseases. Results The crude extract of tissue protein of Metapenaeus ensis showed about 20 bands on SDS-PAGE profile, with relative molecular masses of 12 100 - 105 600. Western blot showed positive reactions of the crude extract with all the 12 serum samples from patients with shrimp allergic diseases. Seven protein bands showed positive reaction with most of the serum samples, of which 4（Mr 36 000, 46 200, 60 000 and 68 000） showed reaction with all the 12 samples. After purification, ELISA proved that the allergic components mainly existed in the elution peak with 0. 1 mol / L NaCl-0. 01 mol / L Tris-HCl of 70% ammonium sulfate precipitation. Conclusion The components with relative molecular masses of 36 000, 46 200, 60 000 and 68 000 were major allergens in tissue protein of Metapenaeus ensis .