本文選題：FOXP3　切入點：cDNA　出處：《第四軍醫(yī)大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 乙型肝炎病毒(hepatitis B virus,HBV)慢性感染是非常嚴(yán)重的人類健康問題,對HBV感染慢性化發(fā)生機(jī)制的研究是尋找新的有效的治療方法的基礎(chǔ)。幾十年來世界各國的科學(xué)家就此進(jìn)行了大量的研究,發(fā)現(xiàn)特異性免疫功能低下是HBV感染慢性化的關(guān)鍵,但是未能闡明免疫功能低下發(fā)生的機(jī)制。目前認(rèn)為維持機(jī)體免疫耐受的主要機(jī)制是中樞耐受和外周耐受,在外周耐受中調(diào)節(jié)性T細(xì)胞(Treg細(xì)胞)具有非常重要的作用,已成為當(dāng)前免疫學(xué)研究的熱點。國內(nèi)外多項研究結(jié)果顯示調(diào)節(jié)性T細(xì)胞與慢性HBV感染者特異性免疫反應(yīng)低下有一定的關(guān)聯(lián)。 國外多個研究小組已證明FOXP3(稱叉頭狀/翼狀螺旋轉(zhuǎn)錄因子)在Treg細(xì)胞上特異性表達(dá),且對Treg細(xì)胞的發(fā)育和功能是必需的。這極大地促進(jìn)了在分子水平上對Treg細(xì)胞的認(rèn)識,但當(dāng)前對于FOXP3是如何調(diào)控Treg細(xì)胞的發(fā)育及其通過何種下游靶基因發(fā)生作用的機(jī)制仍知之甚少。闡明這些問題,對尋找以調(diào)節(jié)性T細(xì)胞為靶點的新的治療自身免疫性疾病、腫瘤和包括慢性乙型病毒性肝炎在內(nèi)的慢性感染性疾病具有重要意義。本實驗在成功克隆人FOXP3 cDNA的基礎(chǔ)上,構(gòu)建了人FOXP3蛋白的原核表達(dá)載體并在大腸桿菌中進(jìn)行了表達(dá),為進(jìn)一步完成對該蛋白的純化及免疫學(xué)功能研究奠定了基礎(chǔ)。 方法:從臍帶血分離的單個核細(xì)胞中提取總RNA,巢式RT-PCR技術(shù)擴(kuò)增FOXP3 cDNA,產(chǎn)物純化后T-A克隆連接至中間載體pMD18-T,利用測序正確質(zhì)粒,構(gòu)建其原核表達(dá)載體pRSET-A-FOXP3,轉(zhuǎn)化大腸桿菌BL-2(1DE3)pLysS,IPTG誘導(dǎo)表達(dá)6×His融合蛋白,表達(dá)產(chǎn)物經(jīng)SDS-PAGE及Western blot檢測及鑒定。 結(jié)果與結(jié)論:經(jīng)序列測定、SDS-PAGE及Western blot等方法證實成功克隆了人FOXP3 cDNA,構(gòu)建了其原核表達(dá)載體,并在大腸桿菌中得到有效表達(dá)。
[Abstract]:Chronic hepatitis B virus infection is a very serious human health problem. The study on the mechanism of chronic HBV infection is the basis of finding new and effective treatment methods. In recent decades, scientists all over the world have done a lot of research, and found that the low specific immune function is the key to chronic HBV infection. At present, it is considered that the main mechanism of maintaining immune tolerance is central tolerance and peripheral tolerance, and the regulatory T cells (Treg cells) play a very important role in peripheral tolerance. Many domestic and foreign studies have shown that regulatory T cells are associated with the low specific immune response of chronic HBV infections. Several foreign research groups have demonstrated that FOXP3 (forkhead / winglike helical transcription factor) is specifically expressed on Treg cells and is necessary for the development and function of Treg cells. This has greatly promoted the understanding of Treg cells at the molecular level. However, little is known about how FOXP3 regulates the development of Treg cells and the mechanism by which downstream target genes play a role. To elucidate these problems, we can find a new treatment for autoimmune diseases targeting regulatory T cells. Tumor and chronic infectious diseases including chronic hepatitis B are of great significance. Based on the successful cloning of human FOXP3 cDNA, the prokaryotic expression vector of human FOXP3 protein was constructed and expressed in Escherichia coli. It lays a foundation for the further study of the purification and immunological function of the protein. Methods: total RNAs were extracted from mononuclear cells isolated from umbilical cord blood. FOXP3 cDNA was amplified by nested RT-PCR. T-A clone was purified and ligated to the intermediate vector pMD18-T, and the correct plasmid was sequenced. The prokaryotic expression vector pRSET-A-FOXP3 was constructed and transformed into Escherichia coli BL-2DE1DE1DE3DE-pLysSS-IPTG to express 6 脳 His fusion protein. The expression product was detected and identified by SDS-PAGE and Western blot. Results and conclusion: the human FOXP3 cDNAs were cloned successfully by sequencing SDS-PAGE and Western blot, and the prokaryotic expression vector was constructed and expressed effectively in Escherichia coli.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉清泉;洪沙;王平忠;王進(jìn);黃長形;;臍帶血、成人外周血中CD4~+CD25~(high)調(diào)節(jié)性T細(xì)胞分布的比較[J];細(xì)胞與分子免疫學(xué)雜志;2006年03期

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本文編號：1601963