B细胞刺激因子受体（BAFF-R）的基因克

发布时间：2018-05-25 05:21

本文选题：BAFF-R + 蛋白表达　；参考：《河北大学》2006年硕士论文

【摘要】： B淋巴细胞刺激因子(BLyS)能调节B细胞的存活、增殖、发育和分化。但是BLyS过量表达会促进B细胞的恶性增殖，导致自身免疫性疾病的发生。BAFF-R是BLyS的专一受体，对于BLyS介导的B细胞存活和成熟是必要的。BAFF-R胞外区与BLyS配体相结合，然后在细胞内特异地与下游接头分子TRAF3相结合，最终激活非经典的NF-κB信号转导途径，从而调节B细胞的一系列功能。而且外源BLyS受体具有清除体内过量BLyS的潜力，所以BAFF-R的功能以及信号转导途径已经成为国内外研究的热点。本论文对BAFF-R的功能进行了以下两方面的研究： 1．钓取了BAFF-R的胞外区cDNA序列，将BAFF-R胞外区以GST融合蛋白的形式进行表达；并且对表达宿主菌和诱导温度进行了优化；然后用Western-Blotting鉴定了所表达蛋白是目的蛋白；随后用亲和层析的方法纯化了BAFF-R胞外区蛋白，其纯度在85％以上；最后用MTT的方法检测到BAFF-R胞外区蛋白对BLyS促RA-MOS细胞的增殖有抑制作用。 2．钓取了BAFF-R胞内区和TRAF3-C(TRAF3 C端稳定区)的cDNA序列，然后利用酵母双杂交的方法研究BAFF-R胞内区和TRAF3相互作用的分子结合域。将BAFF-R胞内区构建到BD质粒上，将TRAF3-C构建到AD质粒上，，两者共转酵母后有强烈的相互作用。然后构建了11个TRAF3的突变体，分别在TRAF3的N端和C端进行缺失突变，随后与BAFF-R胞内区共转酵母。研究结果表明：BAFF-R胞内区N端382—400位氨基酸的螺旋结构区、中间428—463位氨基酸以及C端543—560位18个氨基酸对于TRAF3与BAFF-R胞内区的结合至关重要。本论文对于BAFF-R功能进行了初步的研究和探讨，BAFF-R胞外区蛋白的获得具有治疗自身免疫性疾病的前景；胞内区与TRAF3相互作用分子结合域的确定对于探讨下游信号转导机制具有重要的意义。
[Abstract]:B lymphocyte stimulating factor BLyS can regulate the survival, proliferation, development and differentiation of B cells. However, overexpression of BLyS can promote the malignant proliferation of B cells and lead to the occurrence of autoimmune diseases. BAFF-R is a specific receptor of BLyS. It is necessary for the survival and maturation of B cells mediated by BLyS to bind the extracellular domain of BAFF-R with BLyS ligand. Then it specifically binds with downstream junction molecule TRAF3 in the cell, and finally activates the non-classical NF- 魏 B signal transduction pathway, thus regulating a series of functions of B cells. Moreover, exogenous BLyS receptors have the potential to eliminate excessive BLyS in vivo, so the function and signal transduction pathway of BAFF-R have become a hot topic at home and abroad. In this paper, the functions of BAFF-R are studied as follows: 1. The extracellular region of BAFF-R was sequenced and the extracellular region of BAFF-R was expressed in the form of GST fusion protein, and the expression host bacteria and induction temperature were optimized, then the expressed protein was identified as the target protein by Western-Blotting. The extracellular domain protein of BAFF-R was purified by affinity chromatography and its purity was more than 85%. Finally, BAFF-R extracellular domain protein was detected by MTT to inhibit the proliferation of RA-MOS cells induced by BLyS. 2. The cDNA sequences of the intracellular region of BAFF-R and the stable region of the C-terminal of TRAF3-C(TRAF3 were sequenced. Then the molecular binding domain between the intracellular region of BAFF-R and the interaction of TRAF3 was studied by yeast two-hybrid method. The intracellular region of BAFF-R was constructed into BD plasmid and TRAF3-C was constructed into AD plasmid. Then, 11 TRAF3 mutants were constructed, which were deleted at the N and C ends of TRAF3, respectively, and then co-transformed into yeast with BAFF-R intracellular region. The results showed that the helical structure of N-terminal 382-400 amino acids, the middle 428-463 amino acids and the C-terminal 543-560 amino acids were important for the binding of TRAF3 to BAFF-R. In this paper, we studied the function of BAFF-R and discussed the prospect of obtaining extracellular domain proteins of BAFF-R for the treatment of autoimmune diseases. The determination of molecular binding domain between intracellular region and TRAF3 is of great significance for the study of downstream signal transduction mechanism.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：Q78;R392

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