大鼠IgE依赖组胺释放因子基因克隆表达及其功能探究.docVIP

大鼠IgE依赖组胺释放因子基因克隆表达及其功能探究 　 作者：陈晓湘, 胡旭初, 徐劲, 郑南才, 余新炳 【摘要】 　　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。 【关键词】 大鼠 IgE依赖组胺释放因子 基因克隆 原核表达 肥大细胞 组胺 　　[Abstract] AIM: To clone and express the gene encoding rat IgEdependent histaminereleasing factor (rHRF) and to study the effect of recombinant rHRF to induce histamine release from rat sensitized mast cells. METHODS: The complete coding region of rHRF was cloned and expressed in BL21 cells. The soluble recombinant rHRF was purified. Aliquots of the mast cells obtained from the lungs of OVAimmunized rats were separately stimulated by recombinant rHRF at different concentration. The released histamine was measured by the OPT spectrofluorometric procedure. RESULTS: The cloned DNA fragment showed 97% nucleotide sequence identity and 95% deduced amino acid sequence identity with the mRNA of rat IgEdependent histaminereleasing factor (or translationally controlled tumor protein) in GenBank. The recombinant rHRF showed a Mr of 24 000 in SDSPAGE. The purified rHRF which was independent of the allergen induced histamine releasing from the sensitized mast cells in a dosedependent manner. CONCLUSION: The recombinant rHRF, which showed the effect of inducing histamine release from sensitized mast cells, would play an important role in the allergen diseases. 　　[Keywords]rat; IgEdependent histaminereleasing factor; gene cloning; prokaryotic expression; mast cell; histamine 　　组胺释放因子是一大类具有促进肥大细胞和嗜碱性粒细胞释放组胺等介质的生物活性物质的总称。根据发挥作用时对IgE依赖与否, 可分为IgE非依赖性和IgE依赖性两类。1995年MacDonald等[1]从人巨噬细胞系U937培养上清中鉴定出一种IgE依赖组胺释放因子, 并证实其与人的翻译控制肿瘤蛋白（translationally controlled tumor protein, TCTP）为同一蛋白, 与小鼠TCTP氨