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目的:获得原核表达的可溶性大鼠IgE依赖组胺释放因子(rHRF),并检测其诱发大鼠致敏肥大细胞释放组胺的功能。方法:克隆Wistar大鼠rHRF基因完整编码区序列并在BL21细胞中进行原核表达,纯化的可溶性重组rHRF刺激卵清蛋白变应原致敏的大鼠肺肥大细胞,利用荧光分光光度法测定组胺释放量。结果:测序证实克隆基因与GenBank中大鼠IgE依赖组胺释放因子(又称翻译控制肿瘤蛋白)mRNA序列的一致性为97%,推测的氨基酸序列一致性为95%。SDS-PAGE显示重组rHRF相对分子质量(Mr)约为24000;重组rHRF诱发大鼠致敏肥大细胞释放组胺不依赖变应原,且呈剂量依赖关系。结论:rHRF具有不依赖变应原诱发大鼠致敏肥大细胞释放组胺的功能,可能在Ⅰ型超敏反应过程中发挥重要作用,为进一步研究rHRF的免疫学功能打下基础。
OBJECTIVE: To obtain soluble rat IgE-dependent histamine release factor (rHRF) expressed in prokaryotes and to detect the function of histamine release induced by sensitized mast cells in rats. Methods: The complete coding region of rHRF gene of Wistar rats was cloned and prokaryotic expressed in BL21 cells. Purified soluble recombinant rHRF stimulated ovalbumin allergen - sensitized rat lung mast cells. Fluorescence spectrophotometry was used to determine histamine Release volume. RESULTS: Sequencing confirmed that the identity of the cloned gene was identical to that of the rat IgE-dependent histamine release factor (GenBank) in GenBank, and the putative amino acid sequence identity was 95%. The molecular weight of recombinant rHRF (Mr) was about 24000 by SDS-PAGE. The histamine released by recombinant rHRF-sensitized mast cells did not depend on the allergen and showed a dose-dependent manner. CONCLUSION: rHRF has the function of histamine release independent of allergen-induced sensitized mast cells in rats and may play an important role in the type I hypersensitivity reaction, which lays the foundation for further study on the immunological function of rHRF.