hTSHR胞外区三个片段的原核表达产物免疫活性分析

According to the structure of Extracellular Domain of Human Thyrotropin Receptor (hTSHR) and the distribution trend of thyroid stimulating antibody (TSAb) or thyroid stimulation blocking antibody (TSBAb), the genes which encodes Extracellular Domain of Human Thyrotropin Receptor can be divided into three segments using Molecular Biological techniques. They can be expressed in three segments respectively in prokaryotic system. These three recombinant proteins can react with TSAb or TSBAb in the serum of patients with Graves' disease or idiopathic myxedema. We measure the differences of the level of TSAb and TSBAb between between different segments, select the domain segment which can specific bind to the TSAb and TSBAb in the patients' seurm, and couple hemocyanin with the three fusion protein (hTSHRn、hTSHRm、hTSHRc) which expressed in prokaryotic system. The BALB/c in experimental group will be injected with these combination respectively 5 times with a interval of 15 days each time, and the BALB/c in control group will be injected with normal saline at the same time. After that, we will compare the level of FT3、FT4、TSH、TRAb、TSAb 、TBAb and the Pathological Changes of thyroid tissue in order to identify the immunogenicity of each segment. The experiment is not only a further discussion of the pathogenesis of AITD but also a research for developing specific detection kits.

根据人促甲状腺激素受体(hTSHR) 胞外区(ecd)的结构特点和TSAb或TSBAb的分布趋势，将TSHR胞外区的编码基因划为三段，采用分子生物学技术在原核系统中分三段表达，这些重组蛋白片段分别与Graves'病和特发性粘液性水肿患者血清（即TSAb或TSBAb）反应，测定不同片段中TSAb和TSBAb水平差异，筛选对TSAb和/或TSBAb阳性血清呈现特异性结合的优势片段，并将血蓝蛋白与原核表达的hTSHRn、hTSHRm、hTSHRc融合蛋白耦联，间隔15 d,注入BALB／c小鼠腹腔内共5次，对照组注射生理盐水，比较各组间血清FT3、FT4、TSH、促甲状腺激素受体抗体(TRAb)、促甲状腺激素受体刺激抗体(TSAb)、促甲状腺激素受体阻滞抗体(TBAb)水平及甲状腺组织病理变化进一步明确各片段的免疫原性，为探讨AITD的发病机制和开发TSAb和TSBAb特异性检测试剂盒奠定基础。

根据人促甲状腺激素受体(hTSHR) 胞外区(ecd)的结构特点和TSAb或TSBAb的分布趋势，将TSHR胞外区的编码基因划为三段，采用分子生物学技术在原核系统中分三段表达，这些重组蛋白片段分别与Graves'病和特发性粘液性水肿患者血清（即TSAb或TSBAb）反应，测定不同片段中TSAb和TSBAb水平差异，筛选对TSAb和/或TSBAb阳性血清呈现特异性结合的优势片段（已完成），并将血蓝蛋白与原核表达的hTSHRc融合蛋白耦联，间隔15 d,注入BALB／c小鼠腹腔内共5次，对照组注射生理盐水，比较各组间血清T4、TSH、促甲状腺激素受体抗体TRAb水平及甲状腺组织病理变化，结果显示：与对照组相比，实验组大部分小鼠血清T4水平下降，TRAb呈阳性，甲状腺组织病理无明显变化，表明hTSHRc片段可以刺激小鼠产生TRAb，可能具有TSBAb抗原表位。hTSHRn片段的免疫原性研究正在进行中。