本文選題：BAFF-R + 蛋白表達�。� 參考：《河北大學(xué)》2006年碩士論文

【摘要】： B淋巴細(xì)胞刺激因子(BLyS)能調(diào)節(jié)B細(xì)胞的存活、增殖、發(fā)育和分化。但是BLyS過量表達會促進B細(xì)胞的惡性增殖，導(dǎo)致自身免疫性疾病的發(fā)生。BAFF-R是BLyS的專一受體，對于BLyS介導(dǎo)的B細(xì)胞存活和成熟是必要的。BAFF-R胞外區(qū)與BLyS配體相結(jié)合，然后在細(xì)胞內(nèi)特異地與下游接頭分子TRAF3相結(jié)合，最終激活非經(jīng)典的NF-κB信號轉(zhuǎn)導(dǎo)途徑，從而調(diào)節(jié)B細(xì)胞的一系列功能。而且外源BLyS受體具有清除體內(nèi)過量BLyS的潛力，所以BAFF-R的功能以及信號轉(zhuǎn)導(dǎo)途徑已經(jīng)成為國內(nèi)外研究的熱點。 本論文對BAFF-R的功能進行了以下兩方面的研究： 1．釣取了BAFF-R的胞外區(qū)cDNA序列，將BAFF-R胞外區(qū)以GST融合蛋白的形式進行表達；并且對表達宿主菌和誘導(dǎo)溫度進行了優(yōu)化；然后用Western-Blotting鑒定了所表達蛋白是目的蛋白；隨后用親和層析的方法純化了BAFF-R胞外區(qū)蛋白，其純度在85％以上；最后用MTT的方法檢測到BAFF-R胞外區(qū)蛋白對BLyS促RA-MOS細(xì)胞的增殖有抑制作用。 2．釣取了BAFF-R胞內(nèi)區(qū)和TRAF3-C(TRAF3 C端穩(wěn)定區(qū))的cDNA序列，然后利用酵母雙雜交的方法研究BAFF-R胞內(nèi)區(qū)和TRAF3相互作用的分子結(jié)合域。將BAFF-R胞內(nèi)區(qū)構(gòu)建到BD質(zhì)粒上，將TRAF3-C構(gòu)建到AD質(zhì)粒上，，兩者共轉(zhuǎn)酵母后有強烈的相互作用。然后構(gòu)建了11個TRAF3的突變體，分別在TRAF3的N端和C端進行缺失突變，隨后與BAFF-R胞內(nèi)區(qū)共轉(zhuǎn)酵母。研究結(jié)果表明：BAFF-R胞內(nèi)區(qū)N端382—400位氨基酸的螺旋結(jié)構(gòu)區(qū)、中間428—463位氨基酸以及C端543—560位18個氨基酸對于TRAF3與BAFF-R胞內(nèi)區(qū)的結(jié)合至關(guān)重要。 本論文對于BAFF-R功能進行了初步的研究和探討，BAFF-R胞外區(qū)蛋白的獲得具有治療自身免疫性疾病的前景；胞內(nèi)區(qū)與TRAF3相互作用分子結(jié)合域的確定對于探討下游信號轉(zhuǎn)導(dǎo)機制具有重要的意義。
[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.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：Q78;R392

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